CN112322782A

CN112322782A - Primer group, kit and method for quickly detecting KSHV through polymerase helix reaction

Info

Publication number: CN112322782A
Application number: CN202011043634.7A
Authority: CN
Inventors: 周素娟
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2020-09-28
Filing date: 2020-09-28
Publication date: 2021-02-05
Anticipated expiration: 2040-09-28
Also published as: CN112322782B

Abstract

The invention relates to a primer set, a kit and a method for rapid detection of KSHV by polymerase helical reaction. The nucleotide sequence of the detection primer Ft is shown in SEQ ID NO.1, the nucleotide sequence of the detection primer Bt is shown in SEQ ID NO.2, and the nucleotide sequence of the acceleration primer IF is shown in SEQ ID NO.3 , and the nucleotide sequence of the acceleration primer IB is shown in SEQ ID NO.4. The polymerase helix reaction of the present invention uses 4 primers for KSHV, covering 5 primer regions, which is more specific than the 2 primers of ordinary PCR, and can be equivalent to the specificity of nested PCR. In addition, the present invention can specifically identify the strains of each subtype of KSHV through the specific selection of the conserved region of the KSHV gene and avoid the similar regions of the herpes virus. The presence or absence of the target gene can be judged by the color reaction after amplification, thus completing the qualitative detection of KSHV. The sensitivity of the polymerase helical reaction amplification method is higher than that of ordinary PCR. The operation is simple and fast, and it has a good application prospect for the development of KSHV detection work on the grass-roots site.

Description

Primer group, kit and method for quickly detecting KSHV through polymerase helix reaction

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a primer group, a kit and a method for quickly detecting KSHV (HHV-8) by polymerase helix reaction.

Background

Kaposi's Sarcoma-associated herpesvirus (KSHV), also known as Human herpesvirus-8 (Human herpesvirus Virus 8, HHV-8), is a gamma-herpesvirus. Researchers believe that KSHV is associated with all Kaposi's Sarcomas (KS) and certain lymphoproliferations, such as primary effusion lymphoma and multicentric Castleman's disease. KSHV is generally present in immunodeficient populations such as organ transplants, HIV-1 infected individuals, and KS is also the most common tumor in HIV-1 patients. KSHV is normally in a latent state and by detecting whether KSHV DNA is actively replicating, the subsequent development of KS can be predicted. The method for effectively, quickly and conveniently detecting the KSHV DNA is established, and has important significance for carrying out the detection of the KSHV DNA on the basic level in site.

At present, the KSHV detection method mainly comprises nested PCR, common PCR, immunological antibody detection and the like. The conventional detection method mainly generates antibodies after virus infection and is confirmed by antibody detection, but since the KSHV infection is in a latent stage for a long time, whether the virus is in an active replication stage can be confirmed by nucleic acid detection. In the past, the DNA is usually sequenced after gene fragments are amplified by nested PCR so as to confirm whether KSHV DNA exists, electrophoresis and sequencing are needed after 2 rounds of PCR, 2 days are needed at the fastest speed, the timeliness is poor, and professional people are needed to perform gene analysis by confirming the gene fragments by using software. The immunological detection method such as ELISA method is antibody detection, which has high sensitivity and strong specificity, but the KSHV is in a latent period for a long time, and the detection result of the antibody cannot predict the progress of the KS disease course. At present, although the most widely applied isothermal amplification technology LAMP has the advantages of high sensitivity, strong specificity and the like, the LAMP also has the defects of complex primer design, high reagent price and the like.

Compared with other nucleic acid amplification technologies, the Polymerase Spiral Reaction (PSR) technology only needs one enzyme and one pair of detection primers (only one pair of detection primers can be used for detection, if the acceleration primer is added, the Reaction can be accelerated to shorten the Reaction time), and the detection can be carried out without accurate temperature-changing equipment. For the operator, the operation is simple and does not require a special professional knowledge background to interpret the results. The kit has high specificity and sensitivity compared with common PCR, can be used for rapid high-flux detection on some basic sites, and has good development prospect. Therefore, it is of great significance to establish a method for detecting KSHV by polymerase helix reaction.

Disclosure of Invention

The invention aims to provide a primer group, a kit and a method for quickly detecting KSHV by polymerase helix reaction.

The purpose of the invention can be realized by the following technical scheme:

the first aspect of the invention provides a primer group for quickly detecting KSHV by polymerase helix reaction, which comprises a detection primer Ft and a detection primer Bt, wherein the nucleotide sequence of the detection primer Ft is shown as SEQ ID NO.1, and the nucleotide sequence of the detection primer Bt is shown as SEQ ID NO. 2. The specific nucleotide sequence is shown as follows:

detection primer Ft:

5’-GACACCCCCTGGGAACCAAGCTGCGAGTGCTTGACGATC-3’(SEQ ID NO.1)；

and (3) detecting a primer Bt:

5’-GAACCAAGGGTCCCCCACAGCGCTCAACTGGTCGAGATC-3’(SEQ ID NO.2)。

further, in one embodiment of the present invention, the primer set for rapid detection of KSHV by polymerase chain reaction further comprises an acceleration primer IF and an acceleration primer IB, wherein the nucleotide sequence of the acceleration primer IF is shown in SEQ ID NO.3, and the nucleotide sequence of the acceleration primer IB is shown in SEQ ID NO. 4. The reaction can be accelerated by adding the accelerating primer, and the reaction time is shortened.

The specific nucleotide sequences of the accelerating primer IF and the accelerating primer IB are shown as follows:

an accelerating primer IF: 5'-GCTGATAGGATACAAAGGTACATGG-3' (SEQ ID NO. 3);

accelerating primer IB: 5'-GGTGTTTGAGGGGGTGGT-3' (SEQ ID NO. 4).

Further, the KSHV was from BCBL1 cell strain.

The kit for quickly detecting KSHV by polymerase helix reaction comprises a primer group for quickly detecting KSHV by polymerase helix reaction, and specifically comprises a detection primer Ft and a detection primer Bt, wherein the nucleotide sequence of the detection primer Ft is shown as SEQ ID No.1, and the nucleotide sequence of the detection primer Bt is shown as SEQ ID No. 2.

Further, in one embodiment of the present invention, the concentration of the detection primer Ft and the concentration of the detection primer Bt are both 40. mu.M.

Furthermore, in one embodiment of the present invention, the kit for rapidly detecting KSHV by polymerase chain reaction further comprises an acceleration primer IF and an acceleration primer IB, wherein the nucleotide sequence of the acceleration primer IF is shown in SEQ ID NO.3, and the nucleotide sequence of the acceleration primer IB is shown in SEQ ID NO. 4.

Further, in one embodiment of the present invention, the concentration of the accelerating primer IF and the accelerating primer IB are both 20. mu.M.

Further, in one embodiment of the present invention, the kit for rapidly detecting KSHV by polymerase helix reaction further comprises the following components:

A. 2 × reaction buffer: 40.0mM Tris-HCl, 20.0mM ammonium sulfate, 20.0mM potassium chloride, 16.0mM magnesium sulfate, 0.2% (v/v) Tween 20, 1.4M betaine, 10.0mM dNTPs (reach);

B. bst DNA polymerase;

C. SYBR Green I (2000X).

Further, in one embodiment of the present invention, the Bst DNA polymerase in component B is preferably an aqueous solution of Bst DNA polymerase at a concentration of 8U/. mu.L.

Further, in one embodiment of the present invention, the concentration ratio of SYBR Green I (2000X) in component C is 2000X; preferably prepared by the following method:

20. mu.L of SYBR Green I (10000X) (dissolved in DMSO) was mixed with 80. mu.L of deionized water and diluted 5-fold to obtain the concentration of SYBR Green I (2000X). When in use, 1 μ L SYBR Green I (2000X) is added into the inner side of a tube cover with a reaction system, and the mixture is turned upside down and dyed.

In a third aspect, the invention provides the use of a primer set for rapid detection of KSHV by polymerase helix reaction or a kit for rapid detection of KSHV by polymerase helix reaction for detecting KSHV infection in a human population.

The fourth aspect of the invention provides a method for rapidly detecting KSHV by polymerase helix reaction, which is to perform PSR isothermal amplification reaction by using a detection primer Ft and a detection primer Bt to detect KSHV in a sample to be detected.

Preferably, an accelerating primer IF and an accelerating primer IB are also added during the amplification reaction, and the reaction can be accelerated by adding the accelerating primer, so that the reaction time is shortened.

Further, the method for rapidly detecting KSHV by polymerase helix reaction specifically comprises the following steps:

(1) extracting KSHV DNA of a sample to be detected by using a conventional nucleic acid extraction method as template DNA, and detecting the concentration, purity and integrity of the total DNA;

(2) keeping the temperature in a water bath at 65 ℃ for 60 minutes to carry out polymerase helix amplification reaction; wherein, the polymerase spiral amplification reaction system is a 25 μ L reaction system: 2 x reaction buffer solution 12.5U L, 40U M detection primer Ft, 40U M detection primer Bt each 0.5U L, 20U M acceleration primer IF, 20U M acceleration primer IB each 0.5U L, 8U/U L Bst DNA polymerase 1.0U L, DNA template 1.0U L, deionized water make up to 25L; finally, 1ul of SYBR Green I (2000 x) is added to the inner side of the tube cover;

(3) after the reaction was complete, the dye on the cap was mixed by inversion and then visually observed for color change. If the color is orange yellow, the KSHV in the sample to be detected is negative; if the color is green, the KSHV positivity in the sample to be detected is indicated.

The nucleotide sequence of the detection primer Ft in the step (2) is shown as SEQ ID NO.1, the nucleotide sequence of the detection primer Bt is shown as SEQ ID NO.2, the nucleotide sequence of the acceleration primer IF is shown as SEQ ID NO.3, and the nucleotide sequence of the acceleration primer IB is shown as SEQ ID NO. 4.

The SYBR Green I (2000X) solution in the step (2) is prepared by the following method:

20. mu.L of SYBR Green I (10000X) (dissolved in DMSO) was mixed with 80. mu.L of deionized water and diluted 5-fold to obtain the concentration of SYBR Green I (2000X).

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the reaction system and reaction conditions for KSHV provided by the invention have the advantages of high sensitivity, low cost, no dependence on precise equipment, simple operation steps and capability of quickly carrying out high-flux detection on a basic site.

(2) The method can reduce the detection time to 60 minutes, shortens the detection period compared with the traditional loop-mediated isothermal amplification technology and PCR technology, and has important significance for the development of a novel isothermal amplification technology and the field detection of microorganisms.

(3) Since the polymerase helix reaction uses 4 primers for KSHV, covering 5 primer regions, the specificity is better than that of 2 primers of ordinary PCR, and can be equal to that of nested PCR. In addition, the invention avoids the similar region of the herpes virus by the specific selection of the KSHV gene conserved region, and can specifically identify the strain of each subtype of the KSHV. The presence or absence of the target gene can be judged by the color reaction after amplification, thereby completing the qualitative detection of KSHV. The sensitivity of the polymerase helix reaction amplification method is higher than that of the common PCR. The operation is simple and convenient, the operation is quick, and the detection of the KSHV in the basic site is carried out, so that the method has good application prospect.

(4) The method is used for amplification under the condition of heating to an isothermal condition in a water bath, saves the time of heating and cooling compared with PCR, and is short in time consumption, and result interpretation can be completed within 60 minutes.

(5) The technology does not need special and expensive instruments and reagents, the amplification product does not need gel electrophoresis and gene sequencing, the result can be judged by naked eyes by directly developing with fluorescent dye, the operation is simple, convenient and quick, and the detection cost is lower. The kit and the method are particularly suitable for the field detection of the basic level.

Drawings

FIG. 1 shows the position of a primer used in the PSR method in ORF26, which is a conserved region of the gene. Wherein, the front ends of-Ft and-Bt are added with positive and negative 'N' segments to form a detection primer Ft and a detection primer Bt, and the positions of the 'N' segments are marked in figure 1. The primer front and back have been marked by arrows in their position map.

FIG. 2 is a graph showing the results of electrophoresis in experiments conducted by the conventional PCR method and the PSR method. Wherein, Lane M is DNA Marker; lanes 1-3 are amplification products of general PCR;

lanes

4, 5 are the negative and blank controls (deionized water) for normal PCR, respectively;

lanes

11 and 12 show the results of PSR electrophoresis using the first set of primers (Ft-KSHV-1, Bt-KSHV-1, IF-KSHV-1, and IB-KSHV-1);

lanes

9 and 10 show the results of PSR electrophoresis using the second set of primers (Ft-KSHV-2, Bt-KSHV-2, IF-KSHV-2, and IB-KSHV-2);

lanes

6 and 7 show the results of electrophoresis using the third set of primers (Ft-KSHV-3, Bt-KSHV-3, IF-KSHV-3, and IB-KSHV-3); lane 8 is a blank control for the PSR method.

FIG. 3 is a graph showing the results of detection of KSHV, EBV-1, EBV-2 and HCMV by polymerase helix reaction technique.

FIG. 3(a) is a diagram showing an amplification product of the PSR method after fluorescent staining using SYBR Green I (2000X). From left to right, the color reaction graphs of PSR-method amplification products of a KSHV positive BCBL-1 strain, a KSHV positive BCBL-1 strain complex hole, an EBV 1-type strain, an EBV 2-type strain, an HCMV-gB1 strain, an HCMV-gB3 strain and a negative control are respectively shown.

FIG. 3(b) is a 2% agarose gel electrophoresis chart showing the results of amplification by the PSR method, in which lanes 1 to 7 from left to right are KSHV, KSHV double well, EBV-1 type, EBV-2 type, HCMV, and negative control, respectively. Lane M is DNA Marker; lane 1: a KSHV positive BCBL-1 strain; lane 2: (ii) a KSHV positive BCBL-1 strain counterwell; lane 3: EBV type 1 strains; lane 4: EBV type 2 strains; lane 5: HCMV-gB1 strain; lane 6: HCMV-gB3 strain: lane 7: and (5) negative control.

FIG. 4 is a graph showing the results of the sensitivity test. And (3) performing concentration measurement on the template, performing multiple dilution, and performing amplification detection on the template with different concentrations by using common PCR and PSR methods respectively, so as to determine the sensitivity and detection limit of the PSR method, and comparing the sensitivity with the sensitivity of the traditional method.

FIG. 4(a) shows, from left to right, the color reactions of the PSR amplification products with template concentrations of 70.1 ng/. mu.L, 7.01 ng/. mu.L, 701 pg/. mu.L, 70.1 pg/. mu.L, 7 pg/. mu.L, and 700 fg/. mu.L in 1 to 6 tubes, respectively.

FIG. 4(b) shows a 2% agarose gel electrophoresis of the product of the general PCR method, from left to right, lane M shows a DNA Marker; lanes 1-6 are PCR amplification products with template concentrations of 70.1 ng/. mu.L, 7.01 ng/. mu.L, 701 pg/. mu.L, 70.1 pg/. mu.L, 7 pg/. mu.L, and 700 fg/. mu.L, respectively, and lane 7 is a negative control.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the present invention are commercially available.

Example 1 polymerase helix reaction detection primer screening

1. Design of primers

Three sets of primers as shown in Table 1 were designed for the specific target region conserved region ORF26 of KSHV using online primer design software according to the PSR amplification reaction principle.

TABLE 1 primer information

Primer name sequence (5 'to 3')

Ft-KSHV-1:

GACACCCCCTGGGAACCAAGCTGCGAGTGCTTGACGATC(SEQ ID NO.1)；

Bt-KSHV-1:

GAACCAAGGGTCCCCCACAGCGCTCAACTGGTCGAGATC(SEQ ID NO.2)；

IF-KSHV-1:GCTGATAGGATACAAAGGTACATGG(SEQ ID NO.3)；

IB-KSHV-1:GGTGTTTGAGGGGGTGGT(SEQ ID NO.4)；

Ft-KSHV-2:

GGATTCGAGCACAATGGTGGGACAGCAACACCCAGCTAG(SEQ ID NO.5)；

Bt-KSHV-2:

GGTGGTAACACGAGCTTAGGTAGATGTGGTACACCAACAGC(SEQ ID NO.6)；

IF-KSHV-2:GGCTAAAAAGTGGGGGTAGCA(SEQ ID NO.7)；

IB-KSHV-2:TCAGCAACTGGGGCACG(SEQ ID NO.8)；

Ft-KSHV-3:

ACCATGGGGAACACGATGTCAATGCTACCCCCACTTTTTAGC(SEQ ID NO.9)；

Bt-KSHV-3:

AACTGTAGCACAAGGGGTACCACCCCGGCCGATATTTTGG(SEQ ID NO.10)；

IF-KSHV-3:TCGAGCACAATGGTGGAATCCTTTC(SEQ ID NO.11)；

IB-KSHV-3:GCAGCAGCTGTTGGTGTACCA(SEQ ID NO.12)。

The position diagram of 3 sets of primers Ft, Bt, IF and IB used in the PSR method of the embodiment on the conserved gene region ORF26 is shown in FIG. 1, wherein the front and back "N" segments of-Ft and-Bt are added at the 5' front end of the primer to form a detection primer Ft and a detection primer Bt, and the position of the "N" segment is marked in FIG. 1. The forward and reverse directions of the primers are indicated by arrows in their position diagrams.

2. Method for establishing polymerase helix reaction detection

(1) Reaction system

In Table 1, Ft and Bt concentrations were 40. mu.M, and IF and IB concentrations were 20. mu.M.

2 × reaction stock solution: consists of a mixture of Tris-HCl 40.0mM, ammonium sulfate 20.0mM, potassium chloride 20.0mM, magnesium sulfate 16.0mM, Tween 20 0.2%, betaine 1.4M, dNTPs (research) 10.0 mM.

③ an aqueous solution of Bst DNA Polymerase (Bst DNA Polymerase, Large Fragment, available from NEB) at a concentration of 8U/. mu.L.

(2) Detection method

Firstly, extracting DNA of a KSHV positive BCBL-1 strain of a sample to be detected as template DNA, designing primers aiming at common conserved regions of different subtypes of the KSHV, and screening the designed primers.

Extracting a KSHV positive BCBL-1 strain by adopting a DNA extraction kit, operating according to the kit specification, and determining the concentration and purity of the extracted DNA. Deionized water was used as a blank control.

(vii) polymerase helix amplification reaction of KSHV. Respectively configuring polymerase spiral amplification reaction systems with the total volume of 25 mu L in the reaction tubes; add 2 Xreaction stock solution 12.5. mu.L, detection primer pair Ft and Bt of 40. mu.M 0.5. mu.L, acceleration primers IF and IB of 20. mu.M 0.5. mu.L, Bst DNA polymerase of 1. mu.L, DNA template of 1.0. mu.L, make up volume to 25. mu.L with deionized water. The concentrations of the substances are as follows: Tris-HCl 20.0mM, ammonium sulfate 10.0mM, potassium chloride 10.0mM, magnesium sulfate 8.0mM, Tween 200.1% (v/v), betaine 0.7M, dNTPs (ideal) 5mM, Bst DNA polymerase 8U, detection primers Ft and Bt 0.8. mu.M respectively, and acceleration primers IF and IB 0.4. mu.M respectively. In order to ensure that different screening primer sets can effectively amplify products, the reaction time is specially increased, in the embodiment, the reaction tube is placed in a water bath at 65 ℃ for heat preservation reaction for 80 minutes and then taken out.

The product after the end of amplification was subjected to 2% agarose gel electrophoresis. As shown in FIG. 2, the amplification products of the first set of primers (Ft-KSHV-1, Bt-KSHV-1, IF-KSHV-1, IB-KSHV-1) designed for target KSHV-1 are in

lanes

11 and 12, and the product concentration is extremely high, and several small fragments are accumulated in one place. Lanes of amplification products for the second set of primers (Ft-KSHV-2, Bt-KSHV-2, IF-KSHV-2, IB-KSHV-2) were 9, 10, and the second set of primers was also effective in amplifying the region of interest, but at a relatively lower concentration than the first set. The product lanes of the third set of primers (Ft-KSHV-3, Bt-KSHV-3, IF-KSHV-3, IB-KSHV-3) were 6, 7, and the third set of primers was less effective, had fewer effective bands, and took longer time for the desired reaction than the second and first sets. By comparison, the first set of primers was selected in subsequent experiments and the reaction time was reduced to 60 minutes.

Example 2 detection of KSHV based on polymerase helix reaction isothermal amplification technique and specificity experiments

1. The present embodiment uses other herpesvirus strains as reference control to verify the amplification specificity based on the method for detecting whether the KSHV in the sample to be detected is positive by using polymerase helix reaction isothermal amplification technology. The reagents used were as follows:

a. the detection primers Ft and Bt were used at a concentration of 40. mu.M, and the acceleration primers IF and IB were used at a concentration of 20. mu.M. The primer sequences were as follows (5 'to 3'):

5'-GACACCCCCTGGGAACCAAGCTGCGAGTGCTTGACGATC-3' (SEQ ID NO. 1);

and (3) detecting a primer Bt: 5'-GAACCAAGGGTCCCCCACAGCGCTCAACTGGTCGAGATC-3' (SEQ ID NO. 2);

an accelerating primer IF: 5'-GCTGATAGGATACAAAGGTACATGG-3' (SEQ ID NO. 3);

accelerating primer IB: 5'-GGTGTTTGAGGGGGTGGT-3' (SEQ ID NO. 4).

b.2 × reaction stock: consists of a mixture of Tris-HCl 40.0mM, ammonium sulfate 20.0mM, potassium chloride 20.0mM, magnesium sulfate 16.0mM, Tween 20 0.2% (v/v), betaine 1.4M, dNTPs (research) 10.0 mM;

c. bst DNA Polymerase (Bst DNA Polymerase, Large Fragment, NEB Co.) in an aqueous solution at a concentration of 8U/. mu.L;

d.20. mu.L of SYBR Green I (10000X) (dissolved in DMSO) was mixed with 80. mu.L of deionized water and diluted 5-fold to obtain the concentration of SYBR Green I (2000X). When used, 1. mu.L of SYBR Green I (2000X) was added to the inside of the reaction tube cap after the addition of the system was completed.

2. The KSHV is detected by using the reagent and a polymerase helix reaction amplification technology, and the method comprises the following steps:

(1) extracting DNA of KSHV (HHV8), EBV (HHV4) and HCMV (HHV5) of a sample to be detected: in this embodiment, deionized water is used as a blank control group, wherein KSHV uses multiple wells, EBV uses 2 strains of different subtypes, and HCMV uses 2 strains of different subtypes; and (3) extracting the DNA of each sample by adopting a DNA extraction kit, and determining the concentration and purity of the obtained DNA according to the operation of a kit specification.

(2) Polymerase helix amplification reaction of KSHV: a polymerase spiral amplification reaction system with the total volume of 25 mu L is configured in a reaction tube, 12.5 mu L of 2 multiplied reaction stock solution, 0.5 mu L of 40 mu M detection primers Ft and Bt respectively, 0.5 mu L of 20 mu M acceleration primers IF and IB respectively, 1 mu L of Bst DNA polymerase and 1.0 mu L of DNA template are added, and the volume is supplemented to 25 mu L by deionized water. The concentrations of the substances are as follows: Tris-HCl 20.0mM, ammonium sulfate 10.0mM, potassium chloride 10.0mM, magnesium sulfate 8.0mM, 0.1% (v/v) Tween 20, betaine 0.7M, dNTPs (reach) 5mM, Bst DNA polymerase 8U, primers Ft, Bt each 0.8. mu.M, primers IF, IB each 0.4. mu.M, and 1. mu.L SYBR Green I (2000X) was added to the inside of the reaction tube lid. The reaction tube is placed in a water bath kettle at 65 ℃ for heat preservation reaction for 60 minutes.

(3) And (3) color development detection: after the reaction, the reaction tube was taken out, the fluorescent dye on the cap was mixed by inversion, and the color change was observed with naked eyes.

As a result, as shown in fig. 3, since the drawings cannot provide a color drawing, green and orange are not shown in fig. 3, but actually, the colors of the 1 st and 2 nd tubes are green, the colors of the 3 rd, 4 th, 5 th, 6 th and 7 th tubes are orange, and green and orange are distinguishable in fig. 3. Referring to FIG. 3, from left to right,

tubes

1 and 2 KSHV (HHV8) change color to green, indicating KSHV positivity; EBV (HHV4) in the 3 rd and 4 th tubes, HCMV (HHV5) in the 5 th and 6 th tubes and blank control in the 7 th tube are all orange, and have no color change, which indicates that the 3 rd, 4 th, 5 th, 6 th and 7 th tubes do not contain KSHV, and also indicates that the primer group has good specificity and no cross reaction to other herpes viruses.

Example 3 sensitivity test for KSHV detection by PSR method and comparison of sensitivity by ordinary PCR

The DNA of BCBL-1 strain of KSHV was extracted and tested for concentration and purity, the concentration was found to be 70.1 ng/. mu.L, and 10-fold concentration gradient dilutions were made of the DNA at 7.01 ng/. mu.L, 701 pg/. mu.L, 70.1 pg/. mu.L, 7 pg/. mu.L, and 700 fg/. mu.L, respectively. PSR amplification was performed using deionized water as a negative control, according to the polymerase chain reaction system and reaction conditions constructed in example 1 above. As a result, as shown in fig. 4(a), since the drawings of the specification cannot provide a color drawing, green and orange are not shown in fig. 4(a), but actually, the 1 st, 2 nd, 3 rd, 4 th and 5 th tubes are green in color, the 7 th tube is orange in color, and green and orange are distinguishable in fig. 4 (a). Referring to FIG. 4(a), from left to right, 1-6 tubes were subjected to color reactions of PSR amplification products with template concentrations of 70.1 ng/. mu.L, 7.01 ng/. mu.L, 701 pg/. mu.L, 70.1 pg/. mu.L, 7 pg/. mu.L, and 700 fg/. mu.L, respectively, wherein samples with a concentration of 700 fg/. mu.L failed to be detected by the PSR method. The results show that: the lowest detection limit of the established KSHV polymerase helix reaction method is 7 pg/mu L of KSHV DNA.

The general PCR system is as follows: perfect TM Ex Taq Mix (Dada, Takara) is recommended to be used, the concentration of the upstream primer and the downstream primer is 20 μ M, 25 μ L of the total reaction system is used, the 6 concentrations of the template 70.1ng/μ L, 7.01ng/μ L, 701pg/μ L, 70.1pg/μ L, 7pg/μ L and 700fg/μ L are respectively used for carrying out the experiment, and the reaction system is as follows: 2 PCR mix 12.5ul, forward primer 1ul, reverse primer 1ul, KSHV DNA 1ul at each concentration, supplemented to 25ul using deionized water. The reaction conditions are as follows: 4 minutes at 95 ℃; 94 ℃ for 15 seconds, 55 ℃ for 20 seconds, 72 ℃ for 40 seconds, 40 cycles; the temperature is 72 ℃ for 10 minutes and the temperature is kept at 4 ℃. The results are shown in FIG. 4 (b): the results of 2% agarose gel electrophoresis show: from left to right, lanes 1-6 are PCR amplification products with template concentrations of 70.1 ng/. mu.L, 7.01 ng/. mu.L, 701 pg/. mu.L, 70.1 pg/. mu.L, 7 pg/. mu.L, and 700 fg/. mu.L, respectively, and 7 is a negative control. Of these, the template concentrations at the 2 concentrations of 7 pg/. mu.L and 700 fg/. mu.L were too low to be amplified successfully.

The results show that: the lowest detection limit for KSHV detection by ordinary PCR was 70.1 pg/. mu.L of KSHV DNA.

And (4) conclusion: as can be seen from the above experimental results, the sensitivity of the PCR amplification method is higher than that of the conventional PCR. The PSR method has the following advantages when being used for detection in the basic site:

the operation is simple and convenient: the method has simple operation steps, does not need precise equipment, only needs a common water bath, can directly observe the detection result through the color reaction of the fluorescent dye, saves the steps of electrophoresis and gene sequencing, and can report the result without various software analysis.

And (3) fast: conventional PCR reactions take about 2 hours in a PCR instrument, plus electrophoresis and sequencing takes about at least 2 working days to confirm results. The detection method provided by the invention can obtain results within 60 minutes.

The specificity is strong: because the method uses 4 primers and covers 5 primer regions, the specificity is better than that of 2 primers of common PCR, and the specificity can be equivalent to that of nested PCR. In addition, the invention avoids the similar region of the herpes virus by the specific selection of the KSHV gene conserved region, and can specifically identify the strain of each subtype of the KSHV. The presence or absence of the target gene can be judged by the color reaction after amplification, thereby completing the qualitative detection of KSHV.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggctaaaaag tgggggtagc a 21

<210> 8

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcagcaactg gggcacg 17

<210> 9

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

accatgggga acacgatgtc aatgctaccc ccacttttta gc 42

<210> 10

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aactgtagca caaggggtac caccccggcc gatattttgg 40

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcgagcacaa tggtggaatc ctttc 25

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gcagcagctg ttggtgtacc a 21

Claims

1. A primer group for quickly detecting KSHV by polymerase helix reaction is characterized by comprising a detection primer Ft and a detection primer Bt, wherein the nucleotide sequence of the detection primer Ft is shown as SEQ ID NO.1, and the nucleotide sequence of the detection primer Bt is shown as SEQ ID NO. 2.

2. The primer set for rapid detection of KSHV in polymerase chain reaction according to claim 1, further comprising an accelerating primer IF and an accelerating primer IB, wherein the nucleotide sequence of the accelerating primer IF is shown in SEQ ID No.3, and the nucleotide sequence of the accelerating primer IB is shown in SEQ ID No. 4.

3. A kit for quickly detecting KSHV by polymerase helix reaction is characterized by comprising a detection primer Ft and a detection primer Bt, wherein the nucleotide sequence of the detection primer Ft is shown as SEQ ID NO.1, and the nucleotide sequence of the detection primer Bt is shown as SEQ ID NO. 2.

4. The kit for rapid detection of KSHV in polymerase chain reaction according to claim 3, further comprising an accelerating primer IF and an accelerating primer IB, wherein the nucleotide sequence of the accelerating primer IF is shown in SEQ ID No.3, and the nucleotide sequence of the accelerating primer IB is shown in SEQ ID No. 4.

5. The kit for rapid detection of KSHV according to claim 3 or 4, wherein the concentrations of the detection primer Ft and the detection primer Bt are both 40 μ M, and the concentrations of the acceleration primer IF and the acceleration primer IB are both 20 μ M.

6. A kit for rapid detection of KSHV according to claim 3 or 4, further comprising:

B. bst DNA polymerase;

C. SYBR Green I (2000X).

7. The kit for rapid detection of KSHV according to claim 6, wherein said Bst DNA polymerase in component B is an aqueous solution of Bst DNA polymerase at a concentration of 8U/. mu.L;

the concentration ratio of SYBR Green I (2000X) described in component C was 2000X.

8. Use of the primer set for rapid detection of KSHV by polymerase chain reaction according to claim 1 or 2 or the kit for rapid detection of KSHV by polymerase chain reaction according to claim 3 or 4 for detecting KSHV in a sample.

9. A method for quickly detecting KSHV by polymerase helix reaction is characterized in that a detection primer Ft and a detection primer Bt are utilized, an acceleration primer IF and an acceleration primer IB are added to carry out PSR isothermal amplification reaction, and KSHV in a sample to be detected is detected.

10. The method for rapid detection of KSHV according to claim 9, comprising the steps of:

(2) keeping the temperature in a water bath at 65 ℃ for 60 minutes to carry out polymerase helix amplification reaction; wherein, the polymerase spiral amplification reaction system is a 25 μ L reaction system: 2 x reaction buffer solution 12.5U L, 40U M detection primer Ft, 40U M detection primer Bt each 0.5U L, 20U M acceleration primer IF, 20U M acceleration primer IB each 0.5U L, 8U/U L Bst DNA polymerase 1.0U L, DNA template 1.0U L, deionized water make up to 25L; finally, 1ul of SYBR Green I (2000 ×) was added on the inner side of the reaction lid;

(3) after the reaction is finished, the dye on the cover is reversed and mixed evenly, and then the color change is observed by naked eyes, if the color is orange yellow, the KSHV negativity in the sample to be detected is indicated; if the color is green, the KSHV positivity in the sample to be detected is indicated.