TW202122797A - Methods and kits for detecting dengue virus - Google Patents

Abstract

Disclosed herein is a kit including at least one of four serotype-specific primer sets respectively for use in the detection of dengue virus serotypes 1 to 4 and one species-specific primer set for use in the detection of dengue virus. Also disclosed herein is a method for the detection of the presence of dengue virus in a biological sample using at least one of the primer sets.

Description

Method and kit for detecting dengue fever virus

The present invention relates to a kit for detecting dengue fever virus (DENV). The present invention also relates to a method for detecting DENV present in a biological sample using the kit.

Dengue virus (DENV) is a member of the genus Flavivirus , which is mainly transmitted by the mosquito vector Aedes aegypti. DENV is a positive-sense single-stranded RNA virus (positive-sense single-stranded RNA virus) with a genome of approximately 10.7 kb in length, which can encode and translate three structural proteins (ie, nuclear sheath protein) (nucleocapsid protein) (C), membrane precursor protein (prM), and envelope protein (E)] and seven non-structural (NS) proteins [non-structural (NS) proteins ] (Ie, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). There are currently four antigens-identifiable DENV serotypes (antigenically-distinguishable DENV serotypes) [ie, DENV-1, DENV-2, DENV-3, and DENV-4], and various serotypes are included in the germline ( phylogenetically) different genotypes. It is estimated that more than 300 million people are infected with DENV each year, especially in tropical and subtropical areas such as Africa, Asia, the Pacific, Australia, and the Americas.

Infection of any of these four serotypes can produce a series of clinical diseases, from mild dengue fever (DF) to severe and fatal dengue hemorrhagic fever (DHF) and hemorrhagic shock Syndrome (hemorrhagic shock syndrome, DSS). Symptoms of DENV infection include high fever, severe headache, severe joint, muscle and bone pain, rash, persistent vomiting, severe abdominal pain, and hemorrhagic manifestations, such as ascites, pleura Pleural effusions, bleeding of internal organs, or hemorrhagic shock.

It has been noticed that an infection of a serotype can lead to life-long protection against homotypic reinfection, but there is only temporary cross-protection against heterotypic infection. Studies have shown that heterotypic secondary infections are associated with a high risk of DHF and DSS. Therefore, it is important to provide a method for rapid, sensitive and effective detection or identification of DENV, which is beneficial for patient management and immediate vector control measures, so as to prevent a large-scale epidemic (large-scale epidemic).

Traditional methods for detecting DENV [such as serological diagnosis, as well as identification of culture, physiology or biochemistry] are quite time consuming. Nowadays, molecular techniques used to detect viral genome RNA sequences [for example, relying on reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, qRT -PCR), RT-loop-mediated isothermal amplification (RT-LAMP), etc.] have gradually been accepted as the new standard technology for the detection of DENV.

It has been reported that serotype-specific regions of DENV (such as 3'-untranslated region (3'-UTR)) have been used for diagnosis and epidemiology. The amplification subject of the study. For example, Teoh BT et al. designed a set of 9 DENV-specific primers based on the 3'-UTR of the DENV genome, and these primers can be used for RT-LAMP analysis for testing in clinical practice. All 4 DENV serotypes in the sample and their different genotypes (Teoh BT et al . (2013), BMC Infectious Diseases , 13:387).

In addition, Hu SF et al. revealed four sets of serotype-specific primer sets for RT-LAMP to target different DENV serotypes (each set includes 6 primers), and these primer sets are respectively It is tested by multiple sequence alignments (multiple sequence alignments) of the non-structural protein (NS) 2A of DENV-1, NS4B of DENV-2, NS4A of DENV-3, and 3'-UTR of DENV-4. Design. The test results showed that no cross-reaction of the four serotypes was observed, which means that the designed primer set is suitable for serotyping DENV (Hu SF et al . (2015), BMC Microbiology , 15:265).

Although the above-mentioned literature reports have existed, relevant researchers in the field are still striving to find more satisfactory nucleic acids for rapid and accurate detection of DENV and differentiation of dengue virus serotypes 1 to 4.

Summary of the invention

Thus, in the first aspect, the present invention provides a method for detecting dengue virus (DENV) present in a biological sample, which can reduce at least one of the disadvantages of the prior art. The method includes: Subjecting the nucleic acid in the biological sample to a nucleic acid amplification reaction using a reaction mixture, the reaction mixture including at least one primer set for amplifying a target nucleic acid of dengue virus; and Detecting the presence or absence of an amplified product obtained from the nucleic acid amplification reaction, wherein the presence of the amplified product indicates the presence of dengue virus in the biological sample; The at least one primer group is selected from the group consisting of: (a) A first serotype-specific primer set for amplifying a region in the NS5 gene of dengue fever virus serotype 1 (DENV-1), and the first serotype-specific primer set includes a The first forward outer primer of the nucleotide sequence shown in SEQ ID NO: 1 and the first reverse outer primer of the nucleotide sequence shown in SEQ ID: 2; (b) A second serotype-specific primer set for amplifying a region in the NS5 gene of dengue fever virus serotype 2 (DENV-2), and the second serotype-specific primer set includes a The second forward outer primer of the nucleotide sequence as shown in SEQ ID NO: 7 and a second reverse outer primer having a nucleotide sequence as shown in SEQ ID: 8; (c) A third serotype-specific primer set for amplifying a region in the NS5 gene of dengue fever virus serotype 3 (DENV-3), and the third serotype-specific primer set includes a The third forward outer primer of the nucleotide sequence as shown in SEQ ID NO: 13 and a third reverse outer primer having a nucleotide sequence as shown in SEQ ID: 14; (d) A fourth serotype-specific primer set for amplifying a region of the NS5 gene of dengue fever virus serotype 4 (DENV-4), the fourth serotype-specific primer set including a The fourth forward outer primer of the nucleotide sequence as shown in SEQ ID NO: 19, and a fourth reverse outer primer having a nucleotide sequence as shown in SEQ ID: 20; and (e) A species-specific primer set used to amplify a region of the 3'UTR of the dengue fever virus. The species-specific primer set contains a nucleotide sequence as shown in SEQ ID NO: 25 The fifth forward external primer, and a fifth reverse external primer with a nucleotide sequence as shown in SEQ ID NO: 26.

In the second aspect, the present invention provides a kit for detecting Dengue fever virus (DENV), which can reduce at least one of the disadvantages of the prior art. The set includes at least one of the aforementioned primer sets for amplifying a target nucleic acid of the dengue virus.

Detailed description of the invention

It should be understood that if any previous case publication is quoted here, the previous case publication does not constitute a recognition: in Taiwan or any other country, the previous case publication forms a common general in the art. Part of knowledge.

For the purpose of this specification, it will be clearly understood that the word "comprising" means "including but not limited to", and the word "comprises" has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used in this article have meanings commonly understood by those familiar with the art of the present invention. A person familiar with the art will recognize that many methods and materials similar or equivalent to those described herein can be used to implement the present invention. Of course, the present invention is by no means restricted by the described methods and materials. For clarity, the following definitions are used in this article.

As used herein, the terms "nucleic acid", "nucleic acid sequence" and "nucleotide sequence" refer to a sequence of nucleotides linked by phosphodiester linkages, and may be A deoxyribonucleotide (DNA) or ribonucleotide (RNA) molecule in single or double stranded form. The nucleic acid may include naturally occurring nucleotides (naturally occurring nucleotides) and known analogues (analogues), as well as nucleotides modified on sugar and/or phosphate moieties. This term also includes nucleic acids containing modified backbone residues or linkages. These nucleic acids are synthetic, naturally occurring and non-naturally occurring, and such nucleic acids have It has similar binding properties as the reference nucleic acids, and the nucleic acids are metabolized in a manner similar to the reference nucleotides. As used herein, the term "nucleic acid" and the terms "gene", "cDNA", "mRNA", "oligo-nucleotide" and "polynucleotide" can be used interchangeably .

As used herein, the term "DNA fragment" means a DNA polymer, which is in the form of a separate segment or as a larger DNA construct (DNA polymer). A component of construct, which can be derived from isolated DNA or synthesized chemically or enzymatically, such as by methods disclosed elsewhere.

Unless otherwise specified, in addition to the specific sequences disclosed herein, a nucleic acid sequence also encompasses its complementary sequences, as well as their conservative analogs (conservative analogs), related naturally occurring structural variants and / Or synthetic non-naturally occurring analogues.

Unless otherwise specified, whenever a nucleic acid sequence is indicated, it will be understood that the nucleotides are in the order of 5'to 3'from left to right, and "A" means deoxyadenosine or its Analogs, "C" means deoxycytidine or its analogues, "G" means deoxyguanosine or its analogues, and "T" means deoxythymidine or its analogues Things.

The term "3'" means that a region or position in a polynucleotide or oligonucleotide is more than another region or position in the same polynucleotide or oligonucleotide. 3'[ie, downstream]. The term "5'" means that a region or position in a polynucleotide or oligonucleotide is more 5'than another region or position in the same polynucleotide or oligonucleotide [ie , Upstream]. The terms "3'-end" and "3'-terminus" as used herein for a nucleic acid molecule mean the end of the nucleic acid, which contains a five-carbon The free hydroxyl group on the 3'carbon of the terminal pentose sugar. The terms "5'-end" and "5'-terminus" as used herein for a nucleic acid molecule mean the end of the nucleic acid, which contains a five-carbon attached to the end The free hydroxyl or phosphate group on the 5'carbon of the sugar.

As used herein, the term "primer" means an oligonucleotide with a defined sequence, which is designed to be complementary to a target polynucleotide sequence (complementary), The primer-specific portion hybridizes and undergoes a primer extension reaction. The primer can be used as a starting point for enzymatic polymerization of nucleotides using a polymerase. The length of the primer should be sufficient to prevent itself from annealing to sequences other than the complementary part. Generally, the length of the primer is between 10 and 50 nucleotides. Preferably, the length of the primer is between 13 and 30 nucleotides.

The primers used herein can also be used in nucleic acid amplification reactions. As used herein, the term "amplification" can mean a target sequence [such as a gene or a fragment of a gene] or a number of copies of a complementary sequence of the target sequence. One increase. A "copy" or "amplicon" does not necessarily mean perfect sequence complementarity or identity with the template sequence. For example, the copy can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence changes introduced via a primer that includes a Hybridization but not complementary to the template sequence), and/or sequence errors that occur during the amplification reaction. The products of an amplification reaction are called amplification products.

The nucleic acid amplification reaction can be performed using any method known in the art, for example, a method using multiple heat cycles during the amplification reaction, or a constant temperature The method performed [ie, isothermal amplification]. An example of in vitro amplification that requires thermal cycling is polymerase chain reaction (PCR), in which a sample (such as a biological sample from an individual) is brought into contact with a pair of oligonucleotide primers And under conditions that allow the primers to hybridize to a nucleic acid molecule in the sample. The primers are extended under suitable conditions, separated from the template (dissociated), and then re-annealed, extended, and separated to amplify the number of copies of the nucleic acid molecule. Another example of in vitro amplification reaction technology includes, but is not limited to: PCR, quantitative PCR (qPCR), reverse transcription PCR (reverse transcription PCR, RT-PCR), quantitative RT-PCR (quantitative RT-PCR, RT-qPCR), nested polymerase chain reaction, hot-start polymerase chain reaction, multiplex polymerase chain reaction, ligase chain reaction ligase chain reaction (LCR), gap ligase chain reaction (gLCR), etc.

In another aspect, the isothermal amplification reaction can be performed at a constant temperature, or the amplification reaction process generally occurs at a constant temperature, that is, there is no significant temperature change. Therefore, substantially (substantially) is performed at about the same single temperature. In some examples, the isothermal amplification reaction is substantially isothermal, for example, may include slight temperature changes, such as the temperature change during the amplification reaction does not exceed about 1°C to 3°C.

As used herein, terms such as "target sequence" and "target nucleic acid" are used interchangeably, and mean a specific nucleic acid sequence to be detected and/or amplified. Preferably, the target sequence includes a nucleic acid sequence that hybridizes with the primer in a PCR reaction. The target sequence may also include a probe hybridizing region, which will form a stable hybrid with a detection probe under desired conditions. As recognized by those skilled in the art, a target sequence can be single-stranded or double-stranded. In the content of this disclosure, the target sequence of interest can be in the NS5 gene or the 3'-untranslated region (3'-UTR) of DENV.

As used herein, a "species-specific primer" can hybridize to the target sequence of more than one serotype of DENV.

As used herein, the terms "hybridization" and "annealing" are used interchangeably, and mean that mutually complementary nucleic acid strands react to form a "hybrid" or "double helix" Duplex)" process, which is stabilized by hydrogen bonding between the bases of nucleotide residues. Hydrogen bonds can exist by Watson Crick base pairing, Hoogsteen binding or any other sequence-specific means.

As used herein, terms such as "hybrid" and "duplex" are used interchangeably, and mean a structure formed by the hybridization of two nucleic acids with complementary sequences. This double helix can be formed by complementary binding of two DNA fragments to each other, two RNA fragments complementary binding to each other, or one DNA fragment and one RNA fragment complementary binding. Any one or both members of the double helix may include modified nucleotides and/or nucleotide analogs and nucleoside analogues. As disclosed herein, this double helix is formed by the combination of one or more probes with a sample sequence.

According to the present invention, a method for detecting dengue fever virus (DENV) present in a biological sample is disclosed. The method includes: subjecting the nucleic acid in the biological sample to a nucleic acid amplification reaction using a reaction mixture, the reaction mixture including at least one primer set for amplifying a target nucleic acid of dengue virus; and detecting a nucleic acid obtained from the The presence or absence of amplification products of nucleic acid amplification reactions. The presence of the amplified product indicates the presence of dengue virus in the biological sample.

At least one of the primer sets used to amplify the target nucleic acid of dengue virus includes: (a) A first serotype-specific primer set for amplifying a region in the NS5 gene of dengue fever virus serotype 1 (DENV-1), and the first serotype-specific primer set includes a The first forward outer primer (outer primer) of the nucleotide sequence as shown in the sequence identification number: 1 and the first reverse outer primer having the nucleotide sequence as shown in the sequence identification number: 2; (b) A second serotype-specific primer set for amplifying a region in the NS5 gene of dengue fever virus serotype 2 (DENV-2), and the second serotype-specific primer set includes a The second forward outer primer of the nucleotide sequence as shown in SEQ ID NO: 7 and a second reverse outer primer having a nucleotide sequence as shown in SEQ ID: 8; (c) A third serotype-specific primer set for amplifying a region in the NS5 gene of dengue fever virus serotype 3 (DENV-3), and the third serotype-specific primer set includes a The third forward outer primer of the nucleotide sequence as shown in SEQ ID NO: 13 and a third reverse outer primer having a nucleotide sequence as shown in SEQ ID: 14; (d) A fourth serotype-specific primer set for amplifying a region of the NS5 gene of dengue fever virus serotype 4 (DENV-4), the fourth serotype-specific primer set including a The fourth forward outer primer of the nucleotide sequence as shown in SEQ ID NO: 19, and a fourth reverse outer primer having a nucleotide sequence as shown in SEQ ID: 20; and (e) A species-specific primer set used to amplify a region of the 3'UTR of the dengue fever virus. The species-specific primer set contains a nucleotide sequence as shown in SEQ ID NO: 25 The fifth forward external primer, and a fifth reverse external primer with a nucleotide sequence as shown in SEQ ID NO: 26.

When the species-specific primer set and the serotype-specific primer sets are used in combination, the method of the present invention can simultaneously determine different target sequences in the same sample, especially DENV-specific target sequences [That is, distinguish DENV from Flavivirus (e.g., JEV and ZIKV) and other arboviruses of Alphavirus (e.g., CHIKV)] and different serotypes of DENV Specific target sequence for DENV serotype determination. In order to detect different DENV serotypes, amplification reactions of different target sequences can be performed in separate reaction mixtures or in parallel in the same reaction mixture, and be detected independently.

According to the present invention, the nucleic acid (for example, the viral genome RNA of DENV) from the sample can be extracted from the sample first, and then the amplification reaction can be performed. The operating procedures and conditions for nucleic acid extraction fall within the scope of professionalism and routine techniques of those who are familiar with this technology (for example, see Teoh BT et al. (2013), the same as above and Hu SF et al . (2015) , Same as above).

According to the present invention, the nucleic acid amplification reaction can be performed by using at least one of the following methodologies: polymerase chain reaction (polymerase chain reaction), quantitative polymerase chain reaction (qPCR), reverse transcription Polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR), reverse transcription quantitative polymerase chain reaction (RT-qPCR), nested PCR (nested PCR), hot-start PCR (hot- start PCR), multiplex PCR, in situ PCR, single cell PCR, touchdown PCR, ligase chain reaction (LCR), gap ligation Enzyme chain reaction (gap ligase chain reaction, gLCR) and isothermal amplification reaction (isothermal amplification).

In some specific examples, the nucleic acid amplification reaction is an isothermal amplification reaction using any of various natural or genetically engineered enzymes that can be used for this purpose, for example, using a strong strand-displacement reaction under isothermal conditions. Strand-displacement activity DNA polymerase, thereby eliminating the need for thermal cycling. This polymerase is well known in the art and can include thermophilic bacteria (such as Bacillus stearothermophilus (Bst), Bacillus smithii (Bsm), soil spore spp M (Geobacillus sp. M) ( GspM) DNA polymerase large fragment and hot desulfurized India coli (Thermodesulfatator indicus) (Tin)] of (LF), and variants derived therefrom Engineering (engineered variants), Like Taq DNA polymerase variants (Taq DNA polymerase variants). Non-limiting examples of polymerases that can be used to perform the methods disclosed in the present invention include Bst DNA polymerase (Bst DNA polymerase), Bst 2.0 DNA polymerase (Bst 2.0 DNA polymerase), Bst 2.0 WarmStart™ DNA polymerase (Bst 2.0 WarmStart™ DNA polymerase) (New England Biolabs, Ipswich, Mass.), GspM LF DNA polymerase (GspM LF DNA polymerase), GspSSD LF DNA polymerase (GspSSD LF DNA polymerase), Tin exo-LF DNA polymerase (Tin exo-LF DNA polymerase) and SD DNA polymerase (SD DNA polymerase), Phi29 DNA polymerase (Phi29 DNA polymerase), Bsu DNA polymerase (Bsu DNA polymerase), OmniAmp™ DNA polymerase (OmniAmp™ DNA polymerase) (Lucigen , Middleton, Mich.), Taq DNA polymerase (Taq DNA polymerase), Vent ^® and Deep Vent ^® DNA polymerases (Vent ^® and Deep Vent ^® DNA polymerases) (New England Biolabs), 9°Nm™ DNA polymerase ( 9°Nm™ DNA polymerase) (New England Biolabs), Klenow fragment of DNA polymerase I (Klenow fragment of DNA polymerase I), PhiPRD1 DNA polymerase (PhiPRD1 DNA polymerase), phage M2 DNA polymerase (phage M2 DNA) polymerase), T4 DNA polymerase (T4 DNA polymerase), and T5 DNA polymerase (T5 DNA polymerase), which are commonly used at 50-75°C, and more commonly used at 55-65°C. In some examples, about 1 to 20 U (such as about 1 to 15 U, about 2 to 12 U, about 10 to 20 U, about 2 to 10 U, or about 5 to 10 U) of DNA polymerase is included in In response. In some examples, the polymerase used has strand-displacement activity and lacks 5'-3' exonuclease activity. In a non-limiting example, the DNA polymerase used is Bst 2.0 WarmStart™ DNA polymerase, for example, about 8 U of Bst 2.0 WarmStart™ DNA polymerase per reaction.

Examples of isothermal amplification reactions may include, but are not limited to: strand-displacement amplification (SDA), rolling-circle amplification (RCA), cross-primer amplification Reaction (cross-priming amplification, CPA), nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), helicase-dependent Helicase-dependent amplification (HDA), transcription-mediated amplification (transcription-mediated amplification), loop-mediated amplification (LAMP), and competitive adhesive medium isothermal amplification Reaction (competitive annealing mediated isothermal amplification, CAMP).

When the nucleic acid to be amplified or detected is RNA, a reverse transcription is performed before the isothermal amplification reaction of the target sequence is performed. Reverse transcription can be performed using any suitable reverse transcriptase (reverse transcriptase, RT). RT is well known in the art and can include, but is not limited to: Avian Myeloblastosis Virus (AMV) RT and Moloney Murine Leukemia virus (MMLV) RT. Reverse transcription and DNA amplification reactions can be performed in the same reaction mixture containing the active DNA polymerase and RT enzyme. In addition, the method disclosed in the present invention can use a DNA polymerase with both strong displacement activity and RT activity, such as Pyrophage 3173 DNA polymerase (Pyrophage 3173 DNA polymerase).

In some specific cases, the nucleic acid amplification reaction is performed by RT-LAMP, and the denaturation reaction of the DNA template is not required. Therefore, the LAMP reaction can be performed under isothermal conditions (for example, falling at 60 ℃ to 67 ℃) in the range).

The principle of the LAMP method was revealed in T. Notomi et al. (2000), Nucleic acids research , 28:E63. In short, the reaction is through a pair of "loop-forming" primers [ie, forward inner primer (FIP) and backward inner primer (BIP)] The adhesion and extension reaction (extension) is initiated, followed by a pair of flanking primers [ie, forward outer primer (F3) and backward outer primer (B3)] To perform bonding and extension reactions. The extension response of these primers causes chain-replacement of the loop-forming elements, which fold to form terminal hairpin-loop structures. Once these key structures appear, the amplification reaction method becomes self-sufficient and is performed in a continuous and exponential manner (rather than a cyclic method like PCR) at a constant temperature. Optionally, a pair of additional loop primers [ie, forward loop primer (LF) and backward loop primer (LB)] can be included to accelerate the reaction.

In some specific examples, the first serotype-specific primer set used for LAMP analysis includes the first forward and reverse external primers, and a nucleotide with a sequence identification number: 3 The first forward inner primer of the sequence and a first reverse inner primer having a nucleotide sequence as shown in SEQ ID NO:4. The first serotype-specific primer set may further include one of the following: a first forward loop primer having a nucleotide sequence as shown in sequence identification number: 5, and a first forward loop primer having a nucleotide sequence as shown in sequence identification number: 5; Number: The first reverse loop primer of the nucleotide sequence shown in 6, and their combination, are used to accelerate the LAMP reaction.

In some specific examples, the second serotype-specific primer set used for LAMP analysis includes the second forward and reverse external primers, and a nucleotide with a sequence ID number: 9 The second forward internal primer of the sequence and a second reverse internal primer with a nucleotide sequence as shown in SEQ ID NO:10. The second serotype-specific primer set may further include one of the following: a second forward loop primer with a nucleotide sequence as shown in sequence identification number: 11, and a second forward loop primer with a sequence identification number: 11 Number: The second reverse loop primer of the nucleotide sequence shown in 12, and their combination, are used to accelerate the LAMP reaction.

In some specific examples, the third serotype-specific primer set used for LAMP analysis includes the third forward and reverse external primers, and a nucleotide with a sequence identification number: 15 The third forward internal primer of the sequence and a third reverse internal primer with a nucleotide sequence as shown in SEQ ID NO: 16. The third serotype-specific primer set may further include one of the following: a third forward loop primer with a nucleotide sequence as shown in sequence identification number: 17, and a third forward loop primer with a sequence identification number: 17 No.: The third reverse loop primer of the nucleotide sequence shown in 18, and their combination, are used to accelerate the LAMP reaction.

In some specific examples, the fourth serotype-specific primer set used for LAMP analysis includes the fourth forward and reverse external primers, and a nucleotide with a sequence identification number: 21 The fourth forward inner primer of the sequence and a fourth reverse inner primer with a nucleotide sequence as shown in SEQ ID NO: 22. The fourth serotype-specific primer set may further include one of the following: a fourth forward loop primer with a nucleotide sequence as shown in sequence identification number: 23, and a fourth forward loop primer with a sequence identification number: 23 No.: The fourth reverse loop primer of the nucleotide sequence shown in 24, and their combination, are used to accelerate the LAMP reaction.

In some specific examples, the nucleic acid amplification reaction is performed by CAMP. The principle of the CAMP method is disclosed in Rui Mao, et al. (2018), The Royal Society of Chemistry , 143:639-642. Like LAMP, the CAMP method relies on auto-cycling DNA synthesis using a DNA polymerase with strand displacement activity, and may only require a pair of specifically designed primers, that is, a forward internal primer And a reverse internal primer. For CAMP, additional external primers and loop primer(s) can be designed and selected. They can increase the efficiency and sensitivity of the amplification reaction by accelerating the isothermal amplification reaction and shortening the detection time. The external primer, loop primer, and both added in the CAMP reaction are called O-CAMP, L-CAMP, and OL-CAMP, respectively.

In some specific examples, the species-specific primer set for CAMP includes a fifth forward inner primer having a nucleotide sequence as shown in SEQ ID NO: 27 and a fifth forward inner primer having a sequence ID number: 27 The fifth reverse internal primer of the nucleotide sequence shown in 28. The species-specific primer set for CAMP may further include the fifth forward and reverse external primers and/or a fifth loop primer having a nucleotide sequence as shown in SEQ ID NO: 29.

In some specific examples, the reaction mixture for the LAMP or CAMP reaction further contains a suitable buffer [such as a phosphate buffer or Tris buffer]. The buffer may also include additional components, such as potassium and/or sodium salts (such as KCl or NaCl), magnesium and/or manganese salts (e.g., MgCl ₂ , MgSO ₄ , MnC ₁₂ , and/or Or MnSO ₄ ), and/or ammonium salts [for example, (NH ₄ ) ₂ SO ₄ )], detergents (for example, TWEEN ^® -20, TRITON ^® -X100), or other additives [such as betaine (betaine) or dimethylsulfoxide (dimethylsulfoxide)]. Those skilled in the art can use conventional methods to select an appropriate buffer and any additives. In a non-limiting example, the buffer (pH 8.8) contains 50 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 0.1% TWEEN ^® _{-20, 4} mM MgSO 4, and 0.8 M betaine. Exemplary commercially available reaction buffers include 1×Isothermal Amplification Buffer (New England Biolabs, Ipswich, Mass.), LoopAmp Reaction Mix (LoopAmp Reaction Mix) (Eiken Chemical Co. ., Ltd., Tokyo, Japan), and ILLUMIGENE reaction buffer (Meridian Bioscience, Inc., Cincinnati, Ohio). The reaction mixture also contains nucleotides or nucleotide analogs. In some examples, an isomolar mixture of dATP, dCTP, dGTP, and dTTP (referred to as dNTPs) is included.

In some specific cases, a detectable label can be attached or bound to the primer(s) according to the present invention using techniques well known to those skilled in the art to quantitatively detect the target dengue virus. Examples of detectable labels suitable for the present invention include, but are not limited to: hapten labels, such as biotin and digoxigenin; and a chemiluminescent label , For example, acridinium esters, thioesters, orsulfonamides, luminol, isoluminol, phenanthridinium esters, and the like; A fluorescent label, such as fluorescein, rhodamine, FAM, TET, HEX, JOE, TAMA, NTB, TAMRA, ROX, VIC, NED, cyanin ) Dyes, Texas Red, DABCYL, DABSYL, malachite green, AlexaFluor dyes, LC-Red dyes, PromoFluor dyes, and their derivatives; a radioactive label, for example, ³ H , ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³² P; an enzyme label (enzymatic label), for example, horseradish catalase (horse radish peroxidase), alkaline phosphatase (alkaline phosphatase), and the like; A particle label, such as gold colloids, quantum dots, etc.; and a colorimetric label, such as colored glass or plastic beads. beads) [for example, polystyrene, polypropylene, latex, etc.].

According to the present invention, by adding at least one nucleotide residue to the 5'and/or 3'ends of the primers, or by deleting at least one nucleoside from the 5'and/or 3'ends of the primers Acid residues, so that the primers can be modified to have a length of 15 to 35 nucleotides.

According to the present invention, the amplified product can be detected using at least one of various methods known in the art that can be used for this purpose. For example, the detection method can be turbidity measurement, fluorescence detection, bioluminescence detection, gel electrophoresis, colorimetric detection, Immunoenzymatic detection (immunoenzymatic detection), electrochemical detection (electrochemical detection), and their combination. The test can be semi-quantitative or quantitative. The detection can also be real-time detection, which measures the signal generated by the presence of the amplified product during the nucleic acid amplification reaction to monitor the accumulation of specific amplification products.

When the amplification reaction (such as LAMP) produces a large amount of magnesium pyrophosphate (a white precipitate) and dsDNA, turbidity measurement can be used, which allows visual inspection of the results or the use of a turbidity meter (turbidimeter).

The fluorescence detection can use DNA intercalating dyes, fluorescent molecular beacon probes, or a fluorescent metal indicator (such as calcein).

The bioluminescence detection can measure the stoichiometrically generated coupled conversion of inorganic pyrophosphate during the synthesis of nucleic acid to ATP using the enzyme ATP sulfurylase (ATP sulfurylase). ) Of the bioluminescent output (bioluminescent output).

The colorimetric detection can use a colored indicator for alkaline metal ions, such as hydroxy naphthol blue (HNB) (see Goto et al ., BioTechniques , 2009). Mar, 46(3):167-72) and/or a pH indicator capable of significant color change within the range of pH 7.5 ± 1.1, such as phenol red (pH 6.8-8.2), medium Neutral red (pH 6.8-8.0), cresol red (pH 7.2-8.8), etc.

The electrochemical detection can use a pH meter to directly measure the hydrogen ions released during the amplification reaction (such as LAMP), or use integrated electrodes to measure the electrochemical- The increase in the binding of electrochemically-active DNA-binding redox reporters (such as Methylene Blue) to the amplified product results in a decrease in current.

The immunoenzyme detection includes enzyme-linked immunosorbent assays (ELISA) and lateral flow immunoassays based on the use of specific probes (see, for example, Hashimoto M. et al ., Malar J ., 2018 Jun 19, 17(1):235; Sun Yu-Ling et al ., J. Vet Med Sci., 2014 Apr, 76(4):509–516).

As used herein, the "biological sample" is a sample derived from an organism (such as an animal individual) or components of the organism (such as cells and tissues) . Examples of the biological sample include, but are not limited to: a blood sample, a plasma sample, a serum sample, a corneal tissue sample, and a tear fluid sample. sample), a saliva sample, a cerebrospinal fluid sample, a feces sample, a tissue biopsy, a surgical specimen, a urine Urine sample, a fine needle aspirate, and combinations thereof.

According to the present invention, a kit for detecting dengue fever virus (DENV) is provided, and includes at least one of the above-mentioned primer sets.

In some specific examples, the kit further includes a color indicator for alkaline metal ions, a pH indicator, or a combination thereof. The kit can also further include a nucleic acid dye for monitoring the DNA amplification product. Examples of the nucleic acid dyes include, but are not limited to: ethidium bromide (EtBr), SYBR GREEN I, SYBR GREEN II, SYBR Orange, SYBR GOLD, propidium iodide (Phopidium Iodide, PI), SYTOX Blue, and SYPRO Ruby etc. Detailed description of the preferred embodiment

The present invention will be further described with respect to the following embodiments, but it should be understood that these embodiments are only for illustrative purposes and should not be construed as limitations on the implementation of the present invention. Examples General experimental materials: 1. The primers used in the following experiments are synthesized by Genomics Co., Ltd. 2. The viruses used in the following experiments are listed in Table 1. Table 1 virus Virus strain source Chikungunya virus ( CHIKV) Vaccine strain 181/25 Dr. Scott Weaver (John Sealy Distinguished University Chair in Human Infections and Immunity) Zika virus (Zika virus , ZIKV) MR-766 Professor Cai Jijun [Kaohsiung Medical University (Kaohsiung Medical University), Taiwan) Japanese encephalitis virus (Japanese encephalitis virus, JEV) JE C6/36, msq101021 Centers for Disease Control, Taiwan Dengue virus serotype 1, DENV - 1 Hawaii (NC_001477) American Type Culture Collection (ATCC, Manassas, Va., USA) Dengue virus serotype 2, DENV-2 Thailand 16681 (NC_001474) Dr. Duane J. Gubler (University of Hawaii, Asia-Pacific Institute of Tropical Medicine and Infectious Diseases) Dengue virus serotype 3 ( DENV-3) H87 (NC_001475) ATCC Dengue virus serotype 4 (DENV-4) H241 (NC_002640) ATCC 3. The Vero cells used in the examples were purchased from the American Type Culture Collection Center (ATCC, Manassas, Va., USA). 4. The 1 kb DNA RTU Ladder (250-10 k) (Cat. No. BR341-500, Biomate ^TM ) used in the examples and the 100 bp+3 K DNA RTU ladder (100-3 k) (Cat. No. BR332-500, Biomate ^™ ) are all purchased from Rainbow Biotechnology Co., Ltd. General method: 1. Extraction of RNA:

The viral RNA was extracted using Direct-zolTM RNA MiniPrep (Cat. No. R2052, Unimed Healthcare Inc.) according to the manufacturer's operating instructions. Example 1. A detecting DENV serotypes serotypes 1 to 4, - group-specific primers (serotype-specific primer sets)

NS5 protein is an RNA-dependent RNA polymerase (RNA-dependent RNA polymerase) involved in genome replication. The NS5 genes of all four DENV serotypes are analyzed by the GenBank database maintained by the National Institutes of Health (USA). The conserved region of the referenced NS5 gene is the nucleotide 7839 to 8029 in the full-length genome sequence of DENV-1 (Genbank accession number NC_001477.1), and the full-length genome sequence of DENV-2 (Genbank accession No. NC_001474.2) in nucleotides 9769 to 10011, DENV-3 full-length genome sequence (Genbank accession number NC_001475.2) in nucleotides 7565 to 7881, or DENV-4 full-length genome sequence (Genbank Accession number NC_002640.1) at nucleotides 9549 to 9826.

The primer pairs and loop primer pairs used to detect and distinguish DENV serotypes 1 to 4 are based on the conserved region of the NS5 gene of the respective DENV serotypes using PrimerExplorer V4 software (PrimerExplorer V4 software). Was designed. The specificity of these primer pairs and loop primer pairs is analyzed by BLAST analysis available on the GenBank of the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST).

Therefore, for the specific amplification of various DENV serotypes, a serotype-specific primer set 1 for DENV-1 was prepared, which contains 2 sets of primer pairs ( Named DGI-Opti-B F3 / DGI-Opti-B B3 and DGI-Opti-B FIP / DGI-Opti-B BIP) and 1 set of loop primer pairs (named DGI-Opti-B LF / DGI- Opti-B LB); a serotype-specific primer set 2 for DENV-2, which contains 2 sets of primer pairs (named DGII-Opti-C F3 / DGII-Opti -C B3 and DGII-Opti-C FIP / DGII-Opti-C BIP) and a set of loop primer pairs (named DGII-Opti-C LF / DGII-Opti-C LB); a serum for DENV-3 Type-specific primer set 3 (serotype-specific primer set 3), which contains 2 sets of primer pairs (named DGIII-Opti F3 / DGIII-Opti B3 and DGIII-Opti FIP / DGIII-Opti BIP) and 1 set Loop primer pair (named DGIII-Opti LF / DGIII-Opti LB); and a serotype-specific primer set 4 for DENV-4, which contains 2 sets of primer pairs ( It is named DGIV-Opti F3 / DGIV-Opti B3 and DGIV-Opti FIP / DGIV-Opti BIP) and a set of loop primer pairs (named DGIV-Opti LF / DGIV-Opti LB).

The detailed information of the primer pairs and loop primer pairs described above has been integrated in Table 2-5. a : The nucleotide residue corresponding to the nucleotide shown in the box. b : The nucleotide residue corresponding to the nucleotide indicated on the bottom line. Dengue virus serotype 1 Target pathogen Table 2. Serotype-specific primer set 1 used to detect DENV-1 NS5 gene (NCBI accession number NC_001477.1) Target gene 7964-7983 7900-7883 7939-7968 ^a 8001-7983 ^b 7926-7907 ^a 7858-7879 ^b 8028-8009 7838-7855 Corresponds to nucleotide residues in the target gene Ring primer pair Internal primer pair External primer pair Primer pair DGI-Opti-B LB DGI-Opti-B LF DGI-Opti-B BIP DGI-Opti-B FIP DGI-Opti-B B3 DGI-Opti-B F3 aggatgtattctttacaccacc (SEQ ID NO: 6) tccaggtcctccttttgt (SEQ ID NO: 5) cctagtaaagctatactccggga agggtgt cacatttctcag (SEQ ID NO: 4) gttgccattgggattggttc gaaagtcacagaagtgaaggga (SEQ ID NO: 3) ggagaggactcaccaatatc (SEQ ID NO: 2) tattattgcgctgggctg (SEQ ID NO: 1) Nucleotide sequence (5'→3') a : The nucleotide residue corresponding to the nucleotide shown in the box. b : The nucleotide residue corresponding to the nucleotide indicated on the bottom line. Dengue virus serotype 2 Target pathogen Table 3. Serotype-specific primer set 2 used to detect DENV-2 NS5 gene (NCBI accession number NC_001474.2) Target gene 9908-9925 9847-9831 9888-9906 ^a 9962-9943 ^b 9885-9868 ^a 9805-9821 ^b 9993-10011 9769-9784 Corresponds to nucleotide residues in the target gene Ring primer pair Internal primer pair External primer pair Primer pair DGII-Opti-C LB DGII-Opti-C LF DGII-Opti-C BIP DGII-Opti-C FIP DGII-Opti-C B3 DGII-Opti-C F3 gctcggcagtaccatcac (SEQ ID NO: 12) gggcgtaagacttcccc (SEQ ID NO: 11) gctggcggcaaatgctat t gtatggaccaggttgttcg (SEQ ID NO: 10) gaggtcacgtctgtggaa tggtctttgcgggagac (SEQ ID NO: 9) gttccagactgtcagcatg (SEQ ID NO: 8) ctgattggcagagccc (SEQ ID NO: 7) Nucleotide sequence (5'→3') a : The nucleotide residue corresponding to the nucleotide shown in the box. b : The nucleotide residue corresponding to the nucleotide indicated on the bottom line. Dengue virus serotype 3 Target pathogen Table 4. Serotype-specific primer set 3 used to detect DENV-3 NS5 gene (NCBI accession number NC_001475.2) Target gene 7774-7789 7698-7680 7751-7773 ^a 7854-7834 ^b 7725-7701 ^a 7637-7659 ^b 7881-7864 7565-7582 Corresponds to nucleotide residues in the target gene Ring primer pair Internal primer pair External primer pair Primer pair DGIII-Opti LB DGIII-Opti LF DGIII-Opti BIP DGIII-Opti FIP DGIII-Opti B3 DGIII-Opti F3 ggtcgttcccgaagga (SEQ ID NO: 18) aacccttctttggcttctg (SEQ ID NO: 17) caatggtttgtggaaagaaacat acttttttcagtcctgcacag (SEQ ID NO: 16) gcatgatgtgttgtctctcctc ttt gacctttacaagaaatctgg (SEQ ID NO: 15) ccttttgtgtatcctcgc (SEQ ID NO: 14) ggaacaggctcacaaggt (SEQ ID NO: 13) Nucleotide sequence (5'→3') a : The nucleotide residue corresponding to the nucleotide shown in the box. b : The nucleotide residue corresponding to the nucleotide indicated on the bottom line. Dengue virus serotype 4 Target pathogen Table 5. Serotype-specific primer set 4 used to detect DENV-4 NS5 gene (NCBI accession number NC_002640.1) Target gene 9745-9767 9620-9601 9714-9732 ^a 9790-9773 ^b 9656-9639 ^a 9573-9594 ^b 9826-9809 9549-9564 Corresponds to nucleotide residues in the target gene Ring primer pair Internal primer pair External primer pair Primer pair DGIV-Opti LB DGIV-Opti LF DGIV-Opti BIP DGIV-Opti FIP DGIV-Opti B3 DGIV-Opti F3 catgtagaaaccaggatgaactg (SEQ ID NO: 24) tcccatgtcgttcaagaaga (SEQ ID NO: 23) tttatgaaggatggccgct tgcgagattctggctctc (SEQ ID NO: 22) agatggttcccactgcgg ctagatgagaggtttggcactt (SEQ ID NO: 21) aggcaggctgtttctctt (SEQ ID NO: 20) ggagacgattgcgtgg (SEQ ID NO: 19) Nucleotide sequence (5'→3') Example 2. DENV for detecting a species - specific primer group (species-specific primer set)

The universal primer pairs and loop primers used to detect all four DENV serotypes are designed based on the conserved region of the 3'-UTR of DENV serotypes 1-4 using PrimerExplorer V4 software. The specificity of these universal primer pairs and loop primers was analyzed by BLAST analysis available on the GenBank of the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST). Therefore, a species containing 2 sets of primer pairs (named CAMP-B F2 / CAMP-B B2 and CAMP-B NF / CAMP-B NR) and 1 loop primer (named CAMP-B LFr)- The specific primer set is obtained. The detailed information of the primer pairs and loop primers described above has been integrated in Table 6. a : The nucleotide residue corresponding to the nucleotide shown in the box. b : The nucleotide residue corresponding to the nucleotide indicated on the bottom line. Dengue fever virus Target pathogen Table 6. Species-specific primer sets used to detect DENV 3'-UTR area (NCBI registration number NC_001474.2) Target gene 10634-10615 10639-10659 ^a 10685-10666 ^b 10659-10639 ^a 10578-10600 ^b 10716-10697 10506-10520 Corresponds to nucleotide residues in the target gene Loop primer Internal primer pair External primer pair Primer pair CAMP-B LFr CAMP-B NR CAMP-B NF CAMP-B B2 CAMP-B F2 ccagcgtcaatatgctgttt (SEQ ID NO: 29) gaccagagatcctgctgtct c gttctgtgcctggaatgat (SEQ ID NO: 28) gagacagcaggatctctggtc aaggactagaggttagaggagac (SEQ ID NO: 27) gttgattcaacagcaccatt (SEQ ID NO: 26) gaggagacccctccca (SEQ ID NO: 25) Nucleotide sequence (5'→3') Example 3. serotype according to the present invention - specific primer set for the detection and assessment of DENV serotypes distinguish the utility of 1 to 4

In order to determine the effectiveness of the serotype-specific primer sets 1-4 for the detection and discrimination of the four DENV serotypes, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was performed. experimental method:

After properly culturing the 7 viruses shown in Table 1 in accordance with the supplier’s operating instructions, the cell cultures of each virus thus obtained were used for the RNA analysis described in item 1 of the “General Method”. extraction. The obtained total RNA of each virus was used as a template and used in each set of serotype-specific primer sets 1-4 described in Table 2-5 to perform RT-LAMP. The reaction conditions of this RT-LAMP are shown in Table 7. Table 7 RT-LAMP reaction mixture Volume (µL) RT-LAMP master mix Bst 2.0 DNA polymerase (8 U) (NEB, M0538L) 1 AMV Reverse Transcriptase (2.5 U) (Promega, M5101) 0.25 dNTPs (1.4 mM) 3.5 Minimal Lamp buffer [Contains 50 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 0.1% Tween 20, and 4 mM MgSO ₄ ; pH 8.8] 2.5 Betaine (0.8 M) 2 Phenol red (0.063 mM, pH 8.6-8.8) 0.75 External primer pair F3 (0.1 μM) 0.25 B3 (0.1 μM) 0.25 Internal primer pair FIP (0.8 μM) 2 BIP (0.8 μM) 2 Ring primer pair LF (0.4 μM) 1 LB (0.4 μM) 1 DEPC-treated water 2.5 RNA (1 ng) 1 Operating conditions: an isothermal-amplification reaction was performed at 61°C for 60 minutes, followed by incubation at 80°C for 5 minutes.

The color change of the reaction mixture in the reaction tube is visually observed, and the color of the reaction mixture changes from red to yellow in response to the pH change during the nucleic acid amplification reaction. The resultant product was yellow, indicating that the RNA amplification of DENV was successful. In order to confirm whether the color change is due to the amplification of the target RNA, the obtained product was used for 2% agarose gel electrophoresis (agarose gel electrophoresis) to verify the presence of amplified products (amplicons). result:

Figures 1 to 4 respectively show the detection and discrimination effectiveness of serotype-specific primer sets 1-4 for DENV serotypes 1-4. As shown in Figure 1, gel electrophoresis analysis showed that using serotype-specific primer set 1, a ladder-like banding pattern was observed in the corresponding target virus (ie DENV-1). In addition, a yellow RT-LAMP amplified product was observed with naked eyes in DENV-1. In non-target viruses (ie, DENV-2, DENV-3, DENV-4, and non-DENV viruses), no stepped band patterns and color changes were observed. In addition, similar results were observed for serotype-specific primer sets 2-4 (see Figures 2-4).

These results show that the serotype-specific primer sets 1-4 of the present invention can be used to specifically and effectively detect DENV serotypes 1-4, respectively. Example 4. Evaluation of the universal detection utility of the species- specific primer set according to the present invention for DENV

To determine the universal detection utility of species-specific primer sets for all four DENV serotypes, reverse transcription competitive annealing mediated isothermal amplification (RT-CAMP) was performed. experimental method:

The total RNAs of JEV, ZIKV, CHIKV, DENV-1, DENV-2, DENV-3, and DENV-4 obtained in Example 3 were used as a template and used in the species described in Table 6. Specific primer set for RT-CAMP. The reaction conditions of the RT-CAMP are similar to those described in Table 7, except that the isothermal amplification reaction is performed at 61°C for 90 minutes, and the final concentration of the primers is as follows: CAMP-B F2 (0.2 μM) / CAMP-B B2 (0.2 μM), CAMP-B NF (1.6 μM) / CAMP-B NR (1.6 μM), and CAMP-B LFr (0.8 μM). According to the method described in Example 3, colorimetric assay and agarose gel electrophoresis analysis were performed on the obtained product. result:

Figure 5 shows the universal detection utility of species-specific primer sets for DENV serotypes 1-4. It can be seen from Figure 5 that in all four DENV serotypes, a stepped band pattern and a yellowish RT-CAMP amplified product were observed. In non-target viruses (ie JEV, ZIKV, and CHIKV), no stepped band patterns and color changes were observed.

These results show that the species-specific primer set disclosed in the present invention can be used to universally and effectively detect DENV. Example 5. Test materials for evaluating the utility of the above five primer sets for detecting DENV in plasma samples according to the present invention: A. Preparation of plasma samples:

The plasma samples were obtained from 14 patients (11 males, average age) who were clinically diagnosed with DENV-2 infection and recruited by the National Cheng Kung University Hospital (Tainan, Taiwan). Is 53 years old; 3 women, with an average age of 51 years). The plasma sample was approved by the Institutional Review Board (IRB)/Committee of Medical Ethics and Human Experiments (Tainan, Taiwan) of the Hospital Affiliated to the National Cheng Kung University School of Medicine collect. All procedures involving human subjects comply with the Declaration of Helsinki (Declaration of Helsinki). experimental method:

The plasma samples of each patient were cultured and subcultured once in Vero cells (culture conditions: 37°C and 5% CO ₂ ). The resulting supernatant of each culture was collected, and then the RNA was extracted according to item 1 of the "General Method".

The total RNA (5 ng) of the plasma samples obtained from each patient was used as a template and subjected to RT-LAMP analysis according to the method described in Example 3 for the detection and differentiation of DENV serum Type 1-4.

For comparison, the same experiment was performed using a set of conventional primers described in BT Teoh et al. (2013), BMC Infect. Dis., doi: 10.1186/1471-2334-13-387. The primers of the conventional primer set were synthesized by Genomics Biosci & Tech (Taipei, Taiwan). The detailed information of the introduction group has been integrated in Table 8. a : F3 means forward outer primer, B3 means reverse outer primer, FIP means forward inner primer, BIP means reverse inner primer, and BLP means reverse loop primer. b : The number (1-4) after the slash (/) indicates the DENV serotype (for example, F3/134 indicates the forward external primer for DENV-1, -3, and −4. Dengue fever virus Target pathogen Table 8. The set of conventional introductions described in BT Teoh et al. (2013) 3'UTR area Target gene BLP/1234 BIP/4 BIP/123 FIP/4 FIP/123 B3/4 B3/123 F3/2 F3/134 Primer ^{a, b} Cagagatcctgctgtctc (SEQ ID NO: 38) Acagcatattgacgctgggaragacgctctgtgcctggattgatgttg (SEQ ID NO: 37) Acagcatattgacgctgggaragacgttctgtgcctggaatgatgctg (SEQ ID NO: 36) Aggggtctcctctaaccrctagtcttttttgccacggaagctgtacgc (SEQ ID NO: 35) Aggggtctcctctaaccrctagtctttcaaaccrtggaagctgtacgc (SEQ ID NO: 34) Acctgttggatcaacaacacc (SEQ ID NO: 33) Acctgttgattcaacagcacc (SEQ ID NO: 32) Tgagtaaactatgcagcctgt (SEQ ID NO: 31) Caaaccgtgctgcctgt (SEQ ID NO: 30) Nucleotide sequence (5'→3')

In addition, the total RNA (5 ng) of the plasma samples of each patient was used as a template and the RT-CAMP analysis was performed according to the method described in Example 4 for the general detection of DENV. result:

Table 9 shows the detection utility of the above 6 primer sets for DENV. It can be seen from Table 9 that all plasma samples were detected for DENV by RT-LAMP analysis using serotype-specific primer set 2 or the conventional primer set described in BT Teoh et al. (2013). -2 is positive. In addition, all plasma samples were tested positive for DENV by RT-CAMP analysis using species-specific primer sets. As for the serotype-specific primer sets 1, 3, and 4, none of them can react with DENV-2 RNA, which means that none of these primer sets will cross-react with DENV-2. Table 9 Plasma sample number Serotype-specific primer set Species-specific primer set Acquaintance introductory group 1 2 3 4 1 - + - - + + 2 - + - - + + 3 - + - - + + 4 - + - - + + 5 - + - - + + 6 - + - - + + 7 - + - - + + 8 - + - - + + 9 - + - - + + 10 - + - - + + 11 - + - - + + 12 - + - - + + 13 - + - - + + 14 - + - - + + Detection rate (%) 0 100 0 0 100 100 +: A ladder-shaped band pattern and a yellowish RT-CAMP/RT-LAMP amplified product are observed. -: A ladder-shaped band pattern and a yellow RT-CAMP/RT-LAMP amplification product were not observed.

These results show that the serotype-specific primer sets 1-4 and species-specific primer sets of the present invention can achieve rapid and accurate detection of DENV and distinguish dengue virus serotypes 1 to 4.

All patents and documents cited in this specification are incorporated into this case as reference materials in their entirety. If there is a conflict, the detailed description of the case (including definitions) will prevail.

Although the present invention has been described with reference to the above specific specific examples, it is obvious that many modifications and changes can be made without departing from the scope and spirit of the present invention. Therefore, it is intended that the present invention is only limited by the scope of the patent application attached hereto.

The above and other objectives, features and advantages of the present invention will become apparent with reference to the following detailed description and preferred embodiments and the accompanying drawings, in which: Figure 1 shows the detection of DENV serotype 1 in a reverse transcription loop-mediated isothermal amplification reaction (RT-LAMP) using serotype-specific primer set 1. The above figure is a gel electrophoresis analysis of RT-LAMP reaction products A digital image of the result, where the rubber diameter M=250-10000 bp size mark, and the rubber diameter NTC=No template control (water); and the following figure is a digital image of the colorimetric analysis result using phenol red; Figure 2 shows the detection of DENV serotype 2 with RT-LAMP using serotype-specific primer set 2. The above figure is a digital image of the result of gel electrophoresis analysis of the RT-LAMP reaction product, where the gel diameter M= Markers of 250-10000 bp size, and NTC of rubber diameter = no template control (water); and the following figure is a digital image of the colorimetric analysis result using phenol red; Figure 3 shows the detection of DENV serotype 3 with RT-LAMP using serotype-specific primer set 3. The above figure is a digital image of the result of gel electrophoresis analysis of RT-LAMP reaction products, where the gel diameter M= Markers of 250-10000 bp size, and NTC of rubber diameter = no template control (water); and the following figure is a digital image of the colorimetric analysis result using phenol red; Figure 4 shows the detection of DENV serotype 4 using RT-LAMP of serotype-specific primer set 4. The above figure is a digital image of the result of gel electrophoresis analysis of RT-LAMP reaction products, where the gel diameter M= Markers of 250-10000 bp size, and NTC of rubber diameter = no template control (water); and the figure below is a digital image of the colorimetric analysis result using phenol red; and Figure 5 shows the general detection of DENV in RT-CAMP using species-specific primer sets. The above figure is a digital image of the gel electrophoresis analysis results of RT-CAMP reaction products, where the gel diameter M=100-3000 bp The size of the mark, and the diameter of the glue NTC = no template control (water); and the figure below is a digital image of the colorimetric analysis result using phenol red.

Claims (31)

A method for detecting dengue fever virus (DENV) present in a biological sample, which includes: Subjecting the nucleic acid in the biological sample to a nucleic acid amplification reaction using a reaction mixture, the reaction mixture including at least one primer set for amplifying a target nucleic acid of dengue virus; and Detecting the presence or absence of an amplified product obtained from the nucleic acid amplification reaction, wherein the presence of the amplified product indicates the presence of dengue virus in the biological sample; The at least one primer group is selected from the group consisting of: (a) A first serotype-specific primer set used to amplify a region in the NS5 gene of dengue fever virus serotype 1 (DENV-1), and the first serotype-specific primer set includes a The first forward outer primer of the nucleotide sequence shown in SEQ ID NO: 1 and the first reverse outer primer of the nucleotide sequence shown in SEQ ID: 2; (b) A second serotype-specific primer set for amplifying a region in the NS5 gene of Dengue fever virus serotype 2 (DENV-2), and the second serotype-specific primer set includes a The second forward outer primer of the nucleotide sequence as shown in SEQ ID NO: 7 and a second reverse outer primer having a nucleotide sequence as shown in SEQ ID: 8; (c) A third serotype-specific primer set used to amplify a region of the NS5 gene of dengue fever virus serotype 3 (DENV-3), and the third serotype-specific primer set includes a The third forward outer primer of the nucleotide sequence as shown in SEQ ID NO: 13 and a third reverse outer primer having a nucleotide sequence as shown in SEQ ID: 14; (d) A fourth serotype-specific primer set used to amplify a region of the NS5 gene of dengue fever virus serotype 4 (DENV-4), and the fourth serotype-specific primer set includes a The fourth forward outer primer of the nucleotide sequence as shown in SEQ ID NO: 19, and a fourth reverse outer primer having a nucleotide sequence as shown in SEQ ID: 20; and (e) A species-specific primer set used to amplify a region of the 3'UTR of the dengue virus, the species-specific primer set includes a nucleotide sequence with a sequence identification number: 25 The fifth forward external primer, and a fifth reverse external primer with a nucleotide sequence as shown in SEQ ID NO: 26. The method of claim 1, wherein the nucleic acid amplification reaction is performed by using at least one of the following methodologies: polymerase chain reaction, quantitative polymerase chain reaction (qPCR), reverse transcription polymerase chain reaction (RT -PCR), reverse transcription quantitative polymerase chain reaction (RT-qPCR), nested polymerase chain reaction, hot-start polymerase chain reaction, multiplex polymerase chain reaction, in situ PCR, single-cell PCR, step-down PCR, ligase Chain reaction (LCR), gap ligase chain reaction (gLCR) and isothermal amplification reactions. The method of claim 1, wherein the reaction mixture further comprises a reverse transcriptase and a DNA polymerase. The method of claim 3, wherein the nucleic acid amplification reaction is performed by using a loop-mediated isothermal amplification reaction (LAMP). The method of claim 4, wherein the first serotype-specific primer set further comprises a first forward internal primer having a nucleotide sequence as shown in SEQ ID NO: 3, and a first forward internal primer having a sequence such as Identification Number: The first reverse internal primer of the nucleotide sequence shown in 4. The method of claim 5, wherein the first serotype-specific primer set further includes one of the following: a first forward loop primer having a nucleotide sequence as shown in SEQ ID NO: 5 , A first reverse loop primer with a nucleotide sequence as shown in SEQ ID NO: 6, and their combination. The method of claim 4, wherein the second serotype-specific primer set further includes a second forward internal primer having a nucleotide sequence as shown in SEQ ID NO: 9 and a second forward internal primer having a sequence such as Identification Number: The second reverse internal primer of the nucleotide sequence shown in 10. The method of claim 7, wherein the second serotype-specific primer set further includes one of the following: a second forward loop primer having a nucleotide sequence as shown in SEQ ID NO: 11 , A second reverse loop primer with a nucleotide sequence as shown in SEQ ID NO: 12, and their combination. The method of claim 4, wherein the third serotype-specific primer set further includes a third forward internal primer having a nucleotide sequence as shown in SEQ ID NO: 15 and a third forward internal primer having a sequence such as Identification Number: The third reverse internal primer of the nucleotide sequence shown in 16. The method of claim 9, wherein the third serotype-specific primer set further includes one of the following: a third forward loop primer having a nucleotide sequence as shown in SEQ ID NO: 17 , A third reverse loop primer with a nucleotide sequence as shown in SEQ ID NO: 18, and their combination. The method of claim 4, wherein the fourth serotype-specific primer set further includes a fourth forward internal primer having a nucleotide sequence as shown in SEQ ID NO: 21, and a fourth forward internal primer having a sequence such as Identification Number: The fourth reverse internal primer of the nucleotide sequence shown in 22. The method of claim 11, wherein the fourth serotype-specific primer set further includes one of the following: a fourth forward loop primer having a nucleotide sequence as shown in SEQ ID NO: 23 , A fourth reverse loop primer with a nucleotide sequence as shown in SEQ ID NO: 24, and their combination. The method of claim 3, wherein the species-specific primer set further includes a fifth forward inner primer having a nucleotide sequence as shown in sequence identification number: 27, and a fifth forward inner primer having a sequence identification number such as: The fifth reverse internal primer of the nucleotide sequence shown in 28. The method of claim 13, wherein the nucleic acid amplification reaction is performed by using a competitive adhesive medium isothermal amplification reaction (CAMP). The method of claim 14, wherein the species-specific primer set further includes a fifth loop primer having a nucleotide sequence as shown in SEQ ID NO: 29. The method according to any one of claims 1 to 15, wherein detecting the presence or absence of the amplified product is performed by using at least one of the following methodologies: turbidity measurement, fluorescence detection, bioluminescence detection Detection, gel electrophoresis, colorimetric detection, immunoenzyme detection, electrochemical detection, and their combinations. The method according to any one of claims 1 to 15, wherein at least one of the primers in the at least one primer group is marked with a detectable mark. The method of claim 1, wherein the biological sample is selected from the group consisting of: a blood sample, a plasma sample, a serum sample, a corneal tissue sample, a tear fluid sample, a saliva sample, and a brain Spinal fluid sample, a stool sample, a biopsy, a surgical specimen, a urine sample, a fine needle aspirate, and combinations thereof. A kit for detecting dengue fever virus (DENV), which includes at least one of the following primer sets for amplifying a target nucleic acid of dengue fever virus: (a) A first serotype-specific primer set used to amplify a region in the NS5 gene of dengue fever virus serotype 1 (DENV-1), and the first serotype-specific primer set includes a The first forward outer primer of the nucleotide sequence shown in SEQ ID NO: 1 and the first reverse outer primer of the nucleotide sequence shown in SEQ ID: 2; (b) A second serotype-specific primer set for amplifying a region in the NS5 gene of Dengue fever virus serotype 2 (DENV-2), and the second serotype-specific primer set includes a The second forward outer primer of the nucleotide sequence as shown in SEQ ID NO: 7 and a second reverse outer primer having a nucleotide sequence as shown in SEQ ID: 8; (c) A third serotype-specific primer set used to amplify a region of the NS5 gene of dengue fever virus serotype 3 (DENV-3), and the third serotype-specific primer set includes a The third forward outer primer of the nucleotide sequence as shown in SEQ ID NO: 13 and a third reverse outer primer having a nucleotide sequence as shown in SEQ ID: 14; (d) A fourth serotype-specific primer set used to amplify a region of the NS5 gene of dengue fever virus serotype 4 (DENV-4), and the fourth serotype-specific primer set includes a The fourth forward outer primer of the nucleotide sequence as shown in SEQ ID NO: 19, and a fourth reverse outer primer having a nucleotide sequence as shown in SEQ ID: 20; and (e) A species-specific primer set used to amplify a region of the 3'UTR of the dengue virus, the species-specific primer set includes a nucleotide sequence as shown in the sequence identification number: 25 The fifth forward external primer, and a fifth reverse external primer with a nucleotide sequence as shown in SEQ ID NO: 26. Such as the set of claim 19, wherein at least one of the primers in each primer group is marked with a detectable mark. For example, the set of claim 19, wherein the first serotype-specific primer set further includes a first forward internal primer having a nucleotide sequence as shown in SEQ ID NO: 3, and a first forward internal primer having a nucleotide sequence such as Sequence Identification Number: The first reverse internal primer of the nucleotide sequence shown in 4. Such as the set of claim 21, wherein the first serotype-specific primer set further includes one of the following: a first forward loop having a nucleotide sequence as shown in SEQ ID NO: 5 Primer, a first reverse loop primer with a nucleotide sequence as shown in SEQ ID NO: 6, and their combination. For example, the set of claim 19, wherein the second serotype-specific primer set further includes a second forward internal primer having a nucleotide sequence as shown in SEQ ID NO: 9 and a Sequence Identification Number: The second reverse internal primer of the nucleotide sequence shown in 10. Such as the set of claim 23, wherein the second serotype-specific primer set further includes one of the following: a second forward loop having a nucleotide sequence as shown in SEQ ID NO: 11 Primer, a second reverse loop primer with a nucleotide sequence as shown in SEQ ID NO: 12, and their combination. For example, the set of claim 19, wherein the third serotype-specific primer set further includes a third forward internal primer having a nucleotide sequence as shown in SEQ ID NO: 15 and a third forward internal primer having a nucleotide sequence such as Sequence Identification Number: The third reverse internal primer of the nucleotide sequence shown in 16. Such as the set of claim 25, wherein the third serotype-specific primer set further includes one of the following: a third forward loop having a nucleotide sequence as shown in SEQ ID NO: 17 Primer, a third reverse loop primer with a nucleotide sequence as shown in SEQ ID NO: 18, and their combination. Such as the set of claim 19, wherein the fourth serotype-specific primer set further includes a fourth forward internal primer having a nucleotide sequence as shown in SEQ ID NO: 21, and a fourth forward internal primer having a nucleotide sequence such as Sequence Identification Number: The fourth reverse internal primer of the nucleotide sequence shown in 22. Such as the set of claim 27, wherein the fourth serotype-specific primer set further includes one of the following: a fourth forward loop having a nucleotide sequence as shown in SEQ ID NO: 23 Primer, a fourth reverse loop primer with a nucleotide sequence as shown in SEQ ID NO: 24, and their combination. Such as the set of claim 19, wherein the species-specific primer set further includes a fifth forward inner primer having a nucleotide sequence as shown in sequence identification number: 27, and a fifth forward inner primer having a sequence identification number such as : The fifth reverse internal primer of the nucleotide sequence shown by 28. Such as the set of claim 29, wherein the species-specific primer set further includes a fifth loop primer having a nucleotide sequence as shown in SEQ ID NO: 29. The kit according to any one of claims 19 to 30, further comprising one of the following: a color indicator for alkaline metal ions, a pH indicator, and a combination thereof.

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