CN111662961A - Molecular detection method of alicyclobacillus acidoterrestris - Google Patents

Abstract

The invention discloses a molecular detection method of alicyclobacillus acidoterrestris, under the action of Bca DNA polymerase and RNase H enzyme, at the temperature of 60-65 ℃, the amplification reaction of SPIA can be completed only by one mixed primer. The invention can solve the dependence of the RCA method on the circularized DNA and the false positive phenomenon which is easy to appear in LAMP. This allows SPIA to have higher detection efficiency for RCA and better stability for LAMP. As a new nucleic acid isothermal amplification technology, the SPIA method is more complete and more prominent in the advantages of the isothermal amplification technology.

Description

A kind of molecular detection method of Alicyclobacillus acid soil

technical field

The invention relates to the technical field of microorganism detection, in particular to a molecular detection method of Alicyclobacillus acid soil.

Background technique

In vitro nucleic acid amplification technology is the basis of molecular biology research and an important method of biological analysis. Through nucleic acid amplification, target genes can be amplified and isolated, making them useful in clinical medical diagnosis, gene mutation monitoring, molecular diagnosis, and food safety testing. It has important uses in the fields of environmental safety monitoring and so on.

With the development of molecular biology and the wide application of biophysics in this field, nucleic acid amplification technology has received more and more attention. Common nucleic acid amplification techniques include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) and rolling circle isothermal amplification (RCA) methods, all of which have been widely used in the field of microbial detection and have been successful. It is the most mature detection method so far. But each technology has its own flaws.

As the most primitive in vitro nucleic acid amplification technology, PCR method has the longest development time and the most stable. The method first requires a pair of primers to initiate an amplification reaction, and then repeats the three steps of "denaturation-annealing (renaturation)-extension" to achieve product accumulation. The basic principles of PCR reaction are: ① Denaturation of template DNA: After the template DNA is heated to 90-95 °C for a certain period of time, the double-stranded DNA is dissociated to make it single-stranded so that it can be combined with the primer for the next round of reaction. Preparation; ② Annealing (renaturation) between template DNA and primer: After the template DNA is denatured into a single strand by heating, the temperature is lowered to 50-60 °C, and the primer is paired with the complementary sequence of the template DNA single strand; ③ Primer extension: DNA Under the action of DNA polymerase, the template-primer conjugate uses dNTP as the reaction raw material and the target sequence as the template under the action of DNA polymerase, according to the principle of base pairing and semi-reserved replication, to synthesize a new complementary DNA chain to the template By repeating the three processes of cyclic denaturation-annealing-extension, more "semi-retained replication chains" can be obtained, and this new chain can become the template for the next cycle. It takes 2 to 4 minutes to complete one cycle, and the amplification of the target gene to be amplified can be amplified several million times in 2 to 3 hours. It can be seen that in the process of PCR implementation, constant temperature cycling needs to be performed, which makes the implementation of PCR need to rely on a temperature cycler. The instrument is still expensive so far, which undoubtedly increases the burden for some small and medium-sized enterprises or grass-roots testing institutions, and also increases the difficulty of promoting the method.

The LAMP method was invented by Japanese Notomi in 2000. It is characterized by designing 4 specific primers for 6 regions of the target gene (upstream inner primer FIP, downstream inner primer BIP, upstream outer primer F3 and downstream outer primer B3). , Under the action of strand displacement DNA polymerase (Bst DNA polymerase), 60-65 ℃ constant temperature amplification, 10 ⁹ to 10 ¹⁰ times of nucleic acid amplification can be achieved in about 60 minutes, which has the characteristics of simple operation and high sensitivity. During the operation of LAMP, the problem of false positives is very prominent. According to reports, there are two main reasons for LAMP false positives, one is due to the interaction between multiple primers, and the other is due to the influence of aerosols. But these problems are difficult to really solve in LAMP operation. First, the interaction between primers. Since LAMP itself requires 4 primers, sometimes in order to speed up the reaction rate, as many as 6 primers may be used, so the probability of interaction between primers is even greater. This also adds more difficulty to the primer design process for LAMP. In order to find suitable primers, many sets of primers need to be tested separately to select suitable primers, which is time-consuming, labor-intensive and cost-increasing. In addition, the objective conditions of the laboratory determine the possibility of being affected by aerosols during the LAMP operation. To avoid the influence of aerosols, the laboratory needs to have good conditions, and the operators need to have high quality. These all limit the promotion of LAMP to a certain extent.

The RCA method was started in 1998. This technology simulates the rolling circle replication process of microbial circular DNA in nature. Also, under the action of DNA polymerase (Bst DNA polymerase) with strand displacement activity, a primer can initiate the chain along the circular DNA template. Displacement synthesis to achieve in vitro isothermal amplification of circular DNA templates. For circular DNA, the RCA method has shown better advantages, but for the more common linear DNA, RCA requires a separate step to obtain circularized DNA first. This process relies on padlock probes to obtain circular DNA containing a certain length of the target sequence. Including probe design and the use of probes to generate circular DNA, the RCA reaction takes longer, performs more complicated steps, and costs more.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a molecular detection method for Alicyclobacillus acid soil, which can solve the dependence of RCA method on circularized DNA and the false positive phenomenon that easily occurs in LAMP. This allows SPIA to have higher detection efficiency for RCA and better stability for LAMP. As a new nucleic acid isothermal amplification technology, the SPIA method will be more perfect and highlight the advantages of the isothermal amplification technology, and can solve the problems raised in the above background technology.

To achieve the above object, the present invention provides the following technical solutions:

A molecular detection method for Alicyclobacillus acid soil, under the action of Bca DNA polymerase and RNase H enzyme, at a temperature of 60-65 DEG C, the amplification reaction of SPIA only needs one mixed primer to complete, Specifically include the following steps:

Step 1: At the beginning of the reaction, the mixed primer and the chain termination polynucleotide (blocker) are respectively combined with the corresponding positions of the single-stranded template DNA, and then the complementary chain of the target sequence starts from the 3' end of the primer under the action of Bca DNA polymerase. synthesis;

Step 2: When the chain is extended to the blocker sequence, the chain extension is terminated;

Step 3: When the primer binds to the template and starts to extend, the RNA part at the 5' end of the extension product forms a DNA/RNA hybrid double-strand with the template, which is cleaved and degraded by RNase H enzyme, exposing the RNA part of the primer binding site;

Step 4: After the RNA binding site is exposed, the new free primer will bind to the corresponding position on the template through its RNA part;

Step 5: Until the junction of the blocker, a complete DNA single-strand complementary to the target sequence is replaced; at the same time, the RNA part in the DNA/RNA hybrid double-strand formed by the new primer and the template DNA is again cleaved and degraded by RNase H enzyme, and then Repeat primer binding and strand displacement synthesis to enter the cycle process;

Step 6: The final reaction amplifies a large number of DNA single-stranded products complementary to the target sequence.

Further, the SPIA reaction primer is a combined primer whose 3' end is a DNA fragment and the 5' end is an RNA fragment.

Further, the SPIA reaction requires four deoxynucleoside triphosphates (dNTPs) during nucleic acid amplification, including: deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP). ), deoxyribose thymine triphosphate (dTTP).

Further, in the SPIA reaction, the buffers were 2×Bca DNA polymerase buffer and 5×RNase H buffer reaction buffers carried in the Bca DNA polymerase and RNase H enzyme product set.

Further, the first stage of the SPIA reaction is the pre-denaturation process, adding mixed primers, blocker, dNTPs, MgSO ₄ , Bovine SerumAlbumin, Recombinant RNase Inhibitor, Recombinant RNase Inhibitor, 5×RNase H buffer, 2× The mixture of Bca DNA buffer, genomic DNA and RNase-free water was pre-denatured at 95°C for 80s, then cooled to room temperature, quickly added Bca DNA polymerase and RNase H enzyme, using a real-time fluorescence monitor, set the reaction temperature to 60°C, and the time 120min. During the experiment, the reaction can be terminated at any time according to the intensity of the amplified fluorescent signal.

Compared with the prior art, the beneficial effects of the present invention are:

As a new nucleic acid isothermal amplification technology, the present invention (single-primer isothermal amplification technology for detection of Alicyclobacillus acid soil (SPIA)) is compatible with the currently common polymerase chain reaction (PCR), loop-mediated isothermal amplification Compared with isothermal amplification (LAMP) and rolling circle isothermal amplification (RCA), it has the following advantages:

(1) Single primer: This detection method only needs one primer to complete the entire reaction, which can well avoid non-specific amplification caused by the use of multiple primers.

(2) Avoid RNA interference: SPIA has a unique dependence on RNase H enzyme, which makes it have a direct amplification effect on RNA sequences. Therefore, specific amplification of genomic DNA sequences is performed in the presence of a large amount of mRNA for accurate quantification of gene amount;

(3) The product is single-stranded: the amplified product of this reaction is single-stranded RNA or DNA, and the product can be easily detected by the most conventional detection methods, such as probe hybridization, electrophoresis, and the like.

(4) Real-time monitoring, effective anti-pollution: This detection method can directly judge whether amplification has occurred based on the change of the fluorescent signal. The entire reaction process is carried out in a closed PCR tube, so contamination caused by opening the cap can be effectively avoided, that is, the possibility of aerosol occurrence can be controlled at the source.

Description of drawings

Fig. 1 is the basic principle and process schematic diagram of SPIA reaction of the present invention;

Fig. 2 is SPIA amplification product analysis fluorescence curve diagram of the present invention;

3 is a schematic diagram of ultraviolet lamp irradiation (A) and precipitation observation (B) for analysis of SPIA amplification products of the present invention;

Fig. 4 is SPIA amplification result Fig. 1 of the present invention;

Fig. 5 is SPIA amplification result Fig. 2 of the present invention;

Fig. 6 is SPIA amplification result Fig. 3 of the present invention;

Fig. 7 is SPIA amplification result Fig. 4 of the present invention;

Fig. 8 is SPIA amplification result Fig. 5 of the present invention;

Fig. 9 is SPIA amplification result Fig. 6 of the present invention;

Figure 10 is Figure 7 of the SPIA amplification result of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

A molecular detection method for Alicyclobacillus acid soil, under the action of Bca DNA polymerase and RNase H enzyme, at a temperature of 60-65 DEG C, the amplification reaction of SPIA only needs one mixed primer to complete, Specifically include the following steps:

As shown in Figure 1, at the beginning of the reaction, the mixed primer and the chain termination polynucleotide (blocker) bind to the corresponding positions of the single-stranded template DNA respectively, and then start the complementary chain of the target sequence from the 3' end of the primer under the action of Bca DNA polymerase. When the chain extends to the blocker sequence, the chain extension is terminated; while the primer binds to the template and starts to extend, the RNA part at the 5' end of the extension product forms a DNA/RNA hybrid double-stranded chain with the template, which is cleaved and degraded by RNase H enzyme , exposing the RNA part of the primer binding site; after the RNA binding site is exposed, the new free primer will bind to the corresponding position on the template through its RNA part; because the DNA part of the new primer has a 3'-OH, its strand displacement After the binding of the active DNA polymerase, the DNA primer part at the 5' end of the original primer extension product has more affinity for the template, so the new primer DNA part replaces the DNA part of the original primer and binds to the template and starts strand displacement synthesis; until the blocker junction, displacement A complete DNA single-strand complementary to the target sequence is obtained; at the same time, the RNA part in the DNA/RNA hybrid double-strand formed by the new primer and the template DNA is again cleaved and degraded by RNase H enzyme, and then the primer binding and strand displacement synthesis are repeated. Enter the cycle process; finally, the reaction amplifies a large number of DNA single-stranded products complementary to the target sequence.

The SPIA reaction system includes the following components, see Table 1 below:

Table 1 Composition of SPIA reaction system

1. Primers, the selection of efficient and specific primers is a key factor for the success or failure of nucleic acid amplification in the present invention. The amplification reaction of SPIA requires a mixed primer to complete. First, the mixed primers are complementary to the target sequence and extended under the action of BcaDNA polymerase. With the extension and amplification of Bca DNA polymerase, the formed DNA single strand is continuously replaced, and the amplification reaction continues to cycle, and the amplification product is continuously formed. SPIA amplification has no special requirements for the selection of target sequences, and the primer is a combined primer with a DNA fragment at the 3' end and an RNA fragment at the 5' end. Primer design needs to fully consider its length and composition. Generally, the total length of primers is 10-40nt, and the optimal length is 20-25nt. The optimal length of the DNA part is 7-12nt, and the optimal length of the RNA part is 5-10nt. The composition of primers only needs to conform to the general principles of primer design, such as moderate GC content and Tm value.

Primer Premier 5.0, DNAMAN and other software are generally used for primer design. It is recommended to design multiple pairs of primers for optimal screening to obtain the best amplification effect.

2. Bca DNA polymerase, Bca DNA polymerase is a DNA polymerase with strand displacement activity. Bca DNA polymerase has no 5'→3' exonuclease activity. It has excellent elongation properties and can inhibit DNA secondary structure formation. The optimum temperature for Bca DNA polymerase reaction is 60-65 °C, and when the temperature exceeds 70 °C, Bca DNA polymerase will lose its activity. The amount of Bca DNA polymerase added during the reaction needs to be optimized.

3. dNTPs, the present invention needs four kinds of deoxynucleoside triphosphates (dNTPs) in the process of nucleic acid amplification, including: deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate ( dGTP), deoxyribothymine triphosphate (dTTP). Provides raw material for target DNA sequence amplification. High concentrations of dNTPs will inhibit the amplification reaction, while too low concentrations will reduce product yield.

4. Buffer, in the SPIA reaction system, the buffer can provide a stable pH environment for the reaction system and the reaction of Bca DNA polymerase and RNaseH enzymes. The buffers in the SPIA reaction system are generally the 2×Bca DNA polymerase buffer and 5×RNase H buffer reaction buffers included in the Bca DNA polymerase and RNaseH enzyme product kits.

5. Magnesium ion, in the reaction system of the present invention, the concentration of magnesium ion (Mg ²⁺ ) has a very significant effect on the nucleic acid amplification reaction and is an important parameter. Magnesium ion affects the progress of SPIA reaction from different aspects, such as , it affects the activity of BcaDNA polymerase, thereby affecting the yield of SPIA amplification products. The concentration of magnesium ^ions (Mg ²⁺ ) required by different primer pairs and templates is different. Usually, the concentration of magnesium ions (Mg ²⁺ ) should be optimized to determine the optimum concentration of magnesium ions (Mg ²⁺ ). When a large amount of nucleic acid is synthesized, the pyrophosphate ions precipitated from the deoxynucleoside triphosphate substrates (dNTPs) combine with the magnesium ions (Mg ²⁺ ) in the reaction solution to form a by-product magnesium pyrophosphate white precipitate, so it can be directly The reaction results were qualitatively judged according to whether a white precipitate appeared after centrifugation.

6. Template, the preparation of the template is the initial step of the SPIA reaction, and it is also the most difficult step in the SPIA reaction. The quality of the template preparation directly affects whether the subsequent SPIA reaction can proceed smoothly. There are many kinds of samples that can be used as templates, with a wide range of sources, complex components, and various inhibitors. Therefore, the key to improving the quality of templates is to avoid and remove these inhibitors and increase the yield of templates. The Bca DNA polymerase used in the SPIA reaction is more resistant to inhibitors than the DNA polymerase used in the PCR reaction. Therefore, as long as the purity of the template DNA reaches the purity of the template DNA of the PCR reaction, the template DNA can also be subjected to the SPIA reaction.

The first stage of the SPIA reaction is the pre-denaturation process. Add mixed primers, blocker, dNTPs, MgSO ₄ , Bovine Serum Albumin, Recombinant RNase Inhibitor, Recombinant RNase Inhibitor, 5×RNase H buffer, 2×Bca DNA buffer to a 200μL centrifuge tube , Genomic DNA and RNase-freewater mixture was pre-denatured at 95°C for 80s, then cooled to room temperature, quickly added Bca DNA polymerase and RNase H enzyme, using a real-time fluorescence monitor, set the reaction temperature to 60°C, time 120min, in the experiment During the process, the reaction can be terminated at any time according to the intensity of the amplified fluorescent signal.

After the SPIA reaction is over, the reaction result can be judged in three ways.

Method 1: Fluorescence curve. Using a real-time fluorescence monitor, the amplification of the reaction was determined at any time by watching whether the fluorescence curve peaked (Figure 2). In the negative control, since the template is not amplified, there will be no peaking in the fluorescence curve.

Method 2: Fluorescent dye color development method. The single-stranded DNA obtained in the reaction can be developed with SYBRGreen II (1000×) added to the system. After the dye is combined with single-stranded DNA, light green fluorescence will be generated under the irradiation of ultraviolet light, and the negative result will not generate light green fluorescence because there is no amplification phenomenon (as shown in Figure 3A). Note: 1-negative control; 2-SPIA Amplification product.

Method 3: Precipitation observation method. During the reaction, when the dNTPs are bonded to the nucleic acid chain, pyrophosphate ions are formed, and the pyrophosphate ions combine with the magnesium ions in the reaction system to form an insoluble product, magnesium pyrophosphate, which can be centrifuged after the reaction. (12000r/min, 2min) precipitation, so as to judge the reaction result by observing the formation of precipitation (as shown in Figure 3B), Note: 1-negative control; 2-SPIA amplification product.

The SPIA method is used for the amplification of nucleic acid sequences of Alicyclobacillus acid soil, and the primers are designed as follows:

According to the known sequence of the 16S rRNA genome of Alicyclobacillus acid terrestriali in Genebank (gene number: KC193183.1), homology analysis was performed on the sequence using blast to determine its conserved sequence. Mixed primers and blockers were designed by using Primerpremier 5.0 and DNAMAN software (as shown in Table 2).

901 CATGTGGTTT AATTCGAAGC AACGCGAAGA ACCTTACCAG GGCTTGACAT CCCTCTGACC

961 G GTGCAGAGA TGTACCTTCC CTTCGGGGCA GAGGAGACAG GTGGTGCATG GTTGTCGTCA

1021 GCTCGTGTCG TGAGATGTTG GGTTAAGTCC CGCAACGAGC GCAACCCTTG ATCTGTGTTA

1081 CCAGCACGTAGAGGTGGGGACTCACAGGTGACTGCCCGGCGTAAGTCGGAGGAAGGCGGG .

Table 2 List of primers

The main reagents for SPIA reaction are: Bca BEST enzyme, RNase H enzyme, RNase Inhibitor, MgSO ₄ , dNTPs, Bovine SerumAlbumin, RNase-free water, Genomic DNA Extraction Kit (MiniBESTBacteria Genomic DNA Extraction Kit Ver.3.0), SYBR Green II , medium, mixed primers, chain termination polynucleotides, PCR primers.

The instruments and equipment are: real-time fluorescence monitor (ESE Quant Tube Scanner): ESEGmbh, Stockach, Germany; PCR amplifier (WhatmanT Gradient gene amplifier): Biometra, Germany. JY04S-3E gel imaging system, DYY-10C computer Sanheng multi-purpose electrophoresis instrument power supply, SPX-150B-Z biochemical incubator, QYC-200 constant temperature incubation shaker, ultra-clean workbench, vortex shaker, DrgonMedPipette manual pipetting (0.5～10μL; 10～200μL; 100～1000μL), BL-300-X electronic balance, DSX-280A stainless steel portable autoclave, nucleic acid protein analyzer and other auxiliary equipment.

The SPIA reaction system and reaction conditions are as follows:

(1) Extraction of templates

The preserved Alicyclobacillus acid terrestriali (ATCC49025) was streaked on K's agar medium, cultured at 44°C for 48h, and subcultured twice. A single colony of subculture was picked and inoculated into fresh sterile K's liquid medium, and cultured at 44°C for 24h. The template DNA was extracted using a bacterial DNA extraction kit (purchased from Dalian Bao Biological Co., Ltd.).

(2) Addition of the system

In a 200 μL centrifuge tube, add the reaction system in sequence according to the table below, and the concentrations of each reagent are as follows:

MgSO4 (0.8mmol/L), dNTPs (0.88mmol/L), Bovine Serum Albumin (0.72mg/mL), Recombinant RNase Inhibitor (24U), DNA/RNA mixed primer (8μmol/L), blocker (0.6μmol/L) ), 2×Bca DNA polymerase buffer (2.0μL), 5×RNase H buffer (1.5μL), 1:350SYBR GreenII (1.0μL), template (1.0μL), RNase-free water complement system; template DNA and RNase- Free water was used as positive and negative controls, respectively. (The system added in this step does not contain Bca DNA polymerase, which needs to be added after the pre-denaturation process to prevent the enzyme from being inactivated due to excessive temperature.)

(3) Mixing

The system in step (2) was vortexed on a vortex mixer for 10s, and then centrifuged at a speed of 3000r/min for 10s (the purpose of which is to mix the system and concentrate the liquid on the tube wall to the bottom of the centrifuge tube).

(4) Pre-denaturation

Put the centrifuge tube into the incubator, heat at 95°C for 80s, then quickly place the centrifuge tube on ice for 2min on ice. After the ice bath, Bca DNA polymerase (enzymatic activity 20U) and RNase H enzyme (enzymatic activity 36U) were added.

(5) Mix well

Repeat step (3).

(6) Amplification

The temperature of the real-time fluorescence monitor was set to 60.0 °C, and the reaction time was 90 min.

(7) Observation of reaction results

The amplification of the system was determined by observing whether the fluorescence curve had a peak phenomenon. Positive amplification will have a distinct fluorescence curve.

SPIA amplification results. The SPIA amplification results are shown in FIG. 4 . The present invention only amplified the specific target sequences contained in Salmonella, while for other non-Salmonellas, since they did not contain the target sequences, no amplification reaction occurred. Therefore, the SPIA reaction can specifically amplify target nucleic acid sequences.

Figure 4: Tube1 - negative control; Tube2 - Alicyclobacillus acid terrestrial (ATCC49025); Tube3 - Alicyclobacillus acid terrestriali (No.1); Tube4 - Alicyclobacillus acid terrestriali (No.2) ; Tube5-acid earth alicyclobacillus (No.3); Tube6-acid earth alicyclobacillus (No.4); Tube7-acid earth alicyclobacillus (No.5); Tube8-acid earth Alicyclobacillus (No. 6).

Figure 5: Tube1-negative control; Tube2-Alicyclobacillus acidocaldarius (ATCC49025); Tube3-Alicyclobacillus acidocaldarius (NBRC 111244); Tube4-Alicyclobacillusfastidiosus (DSM 17978); Tube5-Alicyclobacillus acidiphilus (DSM 14558); Tube6-Alicyclobacillus cycloheptanicus (DSM 4006); Tube7-Alicyclobacillus herbarius (DSM 13609); Tube8-Alicyclobacillus sacchari (DSM 17974).

Figure 6: Tube1 - Negative Control; Tube2 - Alicyclobacillus acidophilus (ATCC49025); Tube3 - Acetobacter pasteuri (ATCC 33445); Tube4 - Acetobacter pasteuri (R1); Tube5 - Acetobacter pasteuri (R2) ); Tube6-Acetobacter pasteuri (R3); Tube7-Acetobacter pasteuri (R4); Tube8-Lactobacillus plantarum (CICC21784).

Figure 7: Tube1 - negative control; Tube2 - Alicyclobacillus acid terrestrial (ATCC49025); Tube3 - Lactobacillus plantarum (L1); Tube4 - Lactobacillus plantarum (L2); Tube5 - Lactobacillus plantarum (L3); Tube6 - Lactobacillus plantarum (L4); Tube7-Bacillus cereus (CMCC 63302); Tube8-Bacillus cereus (ATCC11778).

Figure 8: Tube 1 - Negative Control; Tube 2 - Alicyclobacillus terreus (ATCC49025); Tube 3 - Bacillus thuringiensis (CICC 22945); Tube4 - Bacillus subtilis (CICC 10012); Tube5 - Alicyclobacillus thermophilus Acid Bacillus (CICC 20139); Tube 6-Cronobacter (ATCC 51024); Tube 7-Cronobacter (ATCC 51329); Tube 8-Proteus (CMCC 49027).

Figure 9: Tube1 - Negative control; Tube2 - Alicyclobacillus acid terrestriali (ATCC49025); Tube3 - Clostridium butyricum (ATCC 19398); Tube4 - Clostridium butyricum (C1); Tube5 - Clostridium butyricum (C2) ). Tube6 - Clostridium butyricum (C3); Tube7 - Clostridium butyricum (C4); Tube8 - Pediococcus lactis (CICC 20719)

Figure 10: Tube1 - negative control; Tube2 - Alicyclobacillus acid terrestriali (ATCC49025); Tube3 - Pediococcus lactis (P1); Tube4 - Pediococcus lactis (P2); Tube5 - Pediococcus lactis (P3); Tube6 - Lactic acid Pediococcus (P4); Tube7 - Staphylococcus aureus (CICC 21600); Tube8 - Staphylococcus aureus (CMCC 26073).

Based on SPIA, the present invention adds SYBR Green II which can be combined with single-stranded DNA in the reaction system. Compared with traditional EB staining, it not only has low toxicity, no carcinogenicity, but also has higher sensitivity. As a new nucleic acid amplification technology, single-primer isothermal amplification realizes the in vitro amplification of nucleic acid under isothermal conditions, with simple operation, high specificity, high sensitivity and good stability. Compared with the PCR method, SPIA is an isothermal amplification process, and the reaction can be carried out without a special temperature cycler, and the amplification can be completed in an incubator. Compared with LAMP and RCA, the SPIA method has a simpler reaction system, simpler operation and lower cost. And this method can solve the dependence of the RCA method on circularized DNA and the false positive phenomenon that easily occurs in LAMP. This allows SPIA to have higher detection efficiency for RCA and better stability for LAMP. As a new nucleic acid isothermal amplification technology, SPIA method will be more perfect and highlight the advantages of isothermal amplification technology.

The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. The equivalent replacement or change of the inventive concept thereof shall be included within the protection scope of the present invention.

Claims (5)

1. A molecular detection method of alicyclobacillus acidoterrestris is characterized in that under the action of Bca DNA polymerase and RNaseH enzyme, at the temperature of 60-65 ℃, the amplification reaction of SPIA can be completed only by one mixed primer, and the method specifically comprises the following steps:

step 1: when the reaction starts, the mixed primer and the chain termination polynucleotide (blocker) are respectively combined with the corresponding position of the single-stranded template DNA, and then the synthesis of the target sequence complementary chain is started from the 3' end of the primer under the action of Bca DNA polymerase;

step 2: when the chain extends to the blocker sequence, the chain extension is terminated;

and step 3: when the primer is combined with the template and begins to extend, the RNA part at the 5' end of the extension product and the template form a DNA/RNA heterozygosis double chain, and the DNA/RNA heterozygosis double chain is cut and degraded by RNase H enzyme, so that the RNA part combination site of the primer is exposed;

and 4, step 4: after the RNA binding site is exposed, a new free primer will bind to the corresponding position on the template through its RNA portion;

and 5: until the binding position of the blocker, replacing a complete DNA single strand which is complementary with the target sequence; simultaneously, the RNA part in the DNA/RNA heterozygosis double-chain formed by the new primer and the template DNA is cut and degraded by the RNase H enzyme again, then primer combination and strand displacement synthesis are repeatedly carried out, and the cycle process is entered;

step 6: finally, a large amount of DNA single-stranded products which are complementary with the target sequence are amplified through reaction.

2. The method for molecular detection of Alicyclobacillus acidoterrestris according to claim 1, wherein the SPIA reaction primer is a composite primer having a DNA fragment at the 3 'end and an RNA fragment at the 5' end.

3. A method for molecular detection of bacillus acidoterrestris according to claim 1, wherein the SPIA reaction requires 4 deoxynucleoside triphosphates (dNTPs) in the course of performing nucleic acid amplification comprising: deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxyribose thymine triphosphate (dTTP).

4. The molecular detection method of alicyclobacillus acidoterrestris according to claim 1, wherein in SPIA reaction, the buffer is selected from 2 x Bca dnapolymerebuffer and 5 x RNase H buffer reaction buffer carried in the Bca DNA polymerase and RNase H enzyme product set.

5. The molecular detection method of alicyclobacillus acidoterrestris as claimed in claim 1, wherein the first stage of SPIA reaction is a pre-denaturation process, and mixed primers, blocker, dNTPs, MgSO are added into a 200 μ L centrifuge tube₄Bovineseirumulin, recombined RNase Inhibitor, 5 × RNaseH buffer, 2 × Bca DNAbuffer, genomic DNA and RNase-free water mixed solution are pre-denatured for 80s at 95 ℃, then cooled to normal temperature,bca DNA polymerase and RNase H enzyme are quickly added, a real-time fluorescence monitor is utilized, the reaction temperature is set to be 60 ℃, the reaction time is 120min, and the reaction can be stopped at any time according to the amplified fluorescence signal intensity in the test process.

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