CN106591466B - Method for typing vibrio parahaemolyticus based on real-time fluorescent PCR - Google Patents

Abstract

The invention provides a method for typing vibrio parahaemolyticus based on real-time fluorescent PCR, which comprises the following steps: extracting the genome DNA of vibrio parahaemolyticus, and performing fluorescent quantitative PCR detection on the extracted genome DNA, wherein the fluorescent quantitative PCR detection is represented by '1' when a melting curve peak is formed, and the fluorescent quantitative PCR detection is represented by '0' when no melting curve peak is formed; and obtaining a typing result for binary data consisting of 0 and 1. The invention mainly monitors the fluorescence change of a sample to be detected in real time based on the fluorescence quantitative PCR so as to complete the detection of the sample. The method provided by the invention has higher resolution, does not need the step of electrophoresis analysis, which is tedious in operation, and has the advantages of simple and convenient operation and high automation degree.

Description

Method for typing vibrio parahaemolyticus based on real-time fluorescent PCR

Technical Field

The invention belongs to the technical field of microorganism identification, and particularly relates to a vibrio parahaemolyticus typing method, and more particularly relates to a vibrio parahaemolyticus typing method based on real-time fluorescence PCR.

Background

Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a halophilic gram-negative vibrio, which is widely present in sea water and seafood, and food poisoning may be caused by eating food contaminated with the vibrio parahaemolyticus. Vibrio parahaemolyticus has become an important food-borne pathogen in coastal countries such as China, the United states and Japan, and most of bacterial food poisoning is caused by Vibrio parahaemolyticus in coastal provinces of China. With the increase of reports of vibrio parahaemolyticus food poisoning cases, epidemiological investigation on the vibrio parahaemolyticus draws more and more extensive attention of people, and the bacterial typing is an important means for epidemiological research, and has very important scientific significance and application value for judging the genetic relationship among strains, tracing the source of pathogenic bacteria, cutting off the propagation path and making effective countermeasures.

Currently, the typing methods of Vibrio parahaemolyticus can be classified into phenotypic typing and molecular typing. The phenotype typing method has the defects of complex operation, long time consumption, difficult result resolution and the like, and can not meet the requirement. In recent years, with the development of molecular biology techniques, molecular typing techniques such as pulsed-field gel electrophoresis (PFGE), multi-locus sequencing (MLST) and multi-locus variable number tandem repeat analysis (MLVA), randomly amplified polymorphic DNA fragments (RAPD), ribosomal genotyping (ribotyping), arbitrary primer polymerase chain reaction (AP-PCR), amplified fragment length polymorphism analysis (amplified fragment length polymorphism, AFLP), enterobacter intergenic repeat consensus PCR (enteric linked PCR), etc. have been increasingly developed for the purpose of epidemiological control-PCR. These methods have the advantage of being rapid and highly specific compared to phenotyping methods, but they still appear laborious due to the need for an electrophoretic analysis step.

In addition, although the ERIC-PCR method is adopted to amplify the bacterial genome DNA, a DNA fingerprint can be generated, the size and the number of DNA migration bands in a certain range represent the distance and the copy between the repeated sequences, and the classification and identification of pathogenic bacteria can be realized according to the ERIC fingerprint of the genome DNA, the ERIC sequence also exists in highly homologous sequences of the sequences in salmonella and other enterobacteria, so that the defect of low specificity exists when the ERIC sequence is used for carrying out molecular analysis on vibrio parahemolyticus, and vibrio parahemolyticus and microorganisms in the vibrio parahemolyticus can not be distinguished.

In addition, Xiaojian et al reported a new method for genotyping vibrio parahemolyticus gene, and although a gene cluster LVPC consisting of 18 genes is disclosed, since the sites of the selected LVPC gene cluster are subjected to typing analysis, the resolution of the selected gene sites and primers thereof is limited, and the analysis results can be obtained only by analyzing the LVPC gene cluster and the VNTRE gene cluster together. The two sets of gene clusters are analyzed together, so that the operation steps are complicated, the period is long, and the detection workload is large, so that the requirement of rapid typing cannot be met.

Disclosure of Invention

In view of the above, the invention aims to provide a method for typing Vibrio parahaemolyticus based on real-time fluorescent PCR, which has the characteristics of high resolution, simple and convenient operation and rapid typing analysis.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for typing vibrio parahaemolyticus based on real-time fluorescent PCR, which comprises the following steps: extracting the genome DNA of vibrio parahaemolyticus, and performing fluorescent quantitative PCR detection on the extracted genome DNA, wherein the fluorescent quantitative PCR detection is represented by '1' when a melting curve peak is formed, and the fluorescent quantitative PCR detection is represented by '0' when no melting curve peak is formed; and obtaining a typing result for binary data consisting of 0 and 1.

Preferably, the gene detected by the fluorescent quantitative PCR is an LVPC gene cluster; the gene sites of the LVPC gene cluster include VP0383, VP1091, VP1778, VP2902, VPA0074, VPA0716, VP0635, VP1393, VP1563, VPA0895, VPA1336, VPA1708, VP1351, VP2132, VPA0440, VPA1199, VPA1256 and RPI 08.

Preferably, the VP0383 has a nucleotide sequence shown in a sequence table SEQ ID No. 1;

the VP1091 has a nucleotide sequence shown as a sequence table SEQ ID No. 2;

the VP1778 has a nucleotide sequence shown in a sequence table SEQ ID No. 3;

the VP2902 has a nucleotide sequence shown as a sequence table SEQ ID No. 4;

the VPA0074 has a nucleotide sequence shown as a sequence table SEQ ID No. 5;

the VPA0716 has a nucleotide sequence shown in SEQ ID No.6 of the sequence table;

the VP0635 has a nucleotide sequence shown in a sequence table SEQ ID No. 7;

the VP1393 has a nucleotide sequence shown in a sequence table SEQ ID No. 8;

the VP1563 has a nucleotide sequence shown in a sequence table SEQ ID No. 9;

the VPA0895 has a nucleotide sequence shown in a sequence table SEQ ID No. 10;

the VPA1336 has a nucleotide sequence shown as a sequence table SEQ ID No. 11;

the VPA1708 has a nucleotide sequence shown in a sequence table SEQ ID No. 12;

the VP1351 has a nucleotide sequence shown as a sequence table SEQ ID No. 13;

the VP2132 has a nucleotide sequence shown in a sequence table SEQ ID No. 14;

the VPA0440 has a nucleotide sequence shown in a sequence table SEQ ID No. 15;

the VPA1199 has a nucleotide sequence shown in a sequence table SEQ ID No. 16;

the VPA1256 has a nucleotide sequence shown in a sequence table SEQ ID No. 17;

the RPI08 has a nucleotide sequence shown in a sequence table SEQ ID No. 18.

Preferably, the amplified forward primer of VP0383 has a nucleotide sequence shown in SEQ ID No.19 of the sequence table; the amplified reverse primer of VP0383 has a nucleotide sequence shown in SEQ ID No.20 of the sequence table;

the amplified forward primer of VP1091 has a nucleotide sequence shown in SEQ ID No.21 of the sequence table; the reverse primer amplified by the VP1091 has a nucleotide sequence shown in a sequence table SEQ ID No. 22;

the amplified forward primer of VP1778 has the nucleotide sequence shown in SEQ ID No.23 of the sequence table; the reverse primer amplified by VP1778 has the nucleotide sequence shown in SEQ ID No.24 of the sequence table;

the amplified forward primer of VP2902 has a nucleotide sequence shown in SEQ ID No.25 of the sequence table; the reverse primer amplified by VP2902 has a nucleotide sequence shown in a sequence table SEQ ID No. 26;

the forward primer for amplification of VPA0074 has a nucleotide sequence shown in a sequence table SEQ ID No. 27; the reverse primer amplified by the VPA0074 has a nucleotide sequence shown in a sequence table SEQ ID No. 28;

the forward primer amplified by VPA0716 has a nucleotide sequence shown in SEQ ID No.29 of the sequence table; the reverse primer amplified by VPA0716 has a nucleotide sequence shown in SEQ ID No.30 of the sequence table;

the amplified forward primer of VP0635 has the nucleotide sequence shown in SEQ ID No.31 of the sequence table; the amplified reverse primer of VP0635 has the nucleotide sequence shown in SEQ ID No.32 of the sequence table;

the amplified forward primer of VP1393 has a nucleotide sequence shown in SEQ ID No.33 of the sequence table; the amplified reverse primer of VP1393 has a nucleotide sequence shown in SEQ ID No.34 of the sequence table;

the amplified forward primer of VP1563 has a nucleotide sequence shown in SEQ ID No.35 of the sequence table; the reverse primer amplified by the VP1563 has a nucleotide sequence shown in a sequence table SEQ ID No. 36;

the forward primer for amplification of VPA0895 has a nucleotide sequence shown in a sequence table SEQ ID No. 37; the reverse primer amplified by the VPA0895 has a nucleotide sequence shown in a sequence table SEQ ID No. 38;

the forward primer for amplification of VPA1336 has a nucleotide sequence shown in a sequence table SEQ ID No. 39; the reverse primer of the VPA1336 has a nucleotide sequence shown in a sequence table SEQ ID No. 40;

the forward primer for amplification of VPA1708 has a nucleotide sequence shown in a sequence table SEQ ID No. 41; the reverse primer amplified by VPA1708 has a nucleotide sequence shown in a sequence table SEQ ID No. 42;

the amplified forward primer of VP1351 has a nucleotide sequence shown in a sequence table SEQ ID No. 43; the reverse primer amplified by the VP1351 has a nucleotide sequence shown in a sequence table SEQ ID No. 44;

the amplified forward primer of VP2132 has a nucleotide sequence shown in SEQ ID No.45 of the sequence table; the reverse primer amplified by VP2132 has a nucleotide sequence shown in SEQ ID No.46 of the sequence table;

the forward primer for amplification of VPA0440 has a nucleotide sequence shown in a sequence table SEQ ID No. 47; the reverse primer amplified by VPA0440 has a nucleotide sequence shown in a sequence table SEQ ID No. 48;

the forward primer for amplification of VPA1199 has a nucleotide sequence shown in SEQ ID No.49 of the sequence table; the reverse primer of the VPA1199 has a sequence table SEQ ID No. 50;

the forward primer for amplification of the VPA1256 has a nucleotide sequence shown in a sequence table SEQ ID No. 51; the reverse primer amplified by the VPA1256 has a nucleotide sequence shown in a sequence table SEQ ID No. 52;

the amplified forward primer of RPI08 has a nucleotide sequence shown in a sequence table SEQ ID No. 53; the amplified reverse primer of RPI08 has the nucleotide sequence shown in SEQ ID No.54 of the sequence Listing.

Preferably, the LVPC gene cluster gene locus is divided into 3 individual lines to be simultaneously subjected to fluorescent quantitative PCR detection;

the grouping of the 3 systems is as follows: the VP0383, the VP1091, the VP1778, the VP2902, the VPA0074 and the VPA0716 are taken as a first system;

the VP0635, VP1393, VP1563, VPA0895, VPA1336 and VPA1708 are a second system;

the VP1351, VP2132, VPA0440, VPA1199, VPA1256 and RPI08 are in a third system.

Preferably, the PCR reaction conditions for the fluorescent quantitative PCR detection are: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 5s, 30 cycles.

Preferably, the high-resolution melting curve is detected by raising the temperature to 77 ℃ after the last cycle of PCR, and raising the temperature to 93 ℃ at a heating rate of 0.1 ℃/s after 5s of heat preservation.

Preferably, the PCR reaction system for the fluorescent quantitative PCR detection comprises: 1 μ L of LDNA template, 3 μ L of Eva Green dye, 10 μ L of PCR premix, 1 μ L of forward and reverse primers, respectively, and 4 μ L of ultrapure water.

The primers comprise 6 pairs of primers of the same system, and the concentration of the primers is as follows:

a first system: VP 03830.5. mu. mol/L, VP 10910.04. mu. mol/L, VP 17780.12. mu. mol/L, VP 29020.25. mu. mol/L, VPA 00740.26. mu. mol/L, VPA 07160.16. mu. mol/L.

A second system: VP 06350.12. mu. mol/L, VP 13930.6. mu. mol/L, VP 15630.15. mu. mol/L, VPA 08950.18. mu. mol/L, VPA 13360.12. mu. mol/L, VPA 17080.18. mu. mol/L.

And a third system: VP 13510.02. mu. mol/L, VP 21320.4. mu. mol/L, VPA 04400.1. mu. mol/L, VPA 11990.1. mu. mol/L, VPA 12560.26. mu. mol/L, RPI 080.4. mu. mol/L.

The invention provides a method for typing vibrio parahaemolyticus based on real-time fluorescent PCR, which comprises the following steps: extracting the genome DNA of vibrio parahaemolyticus, and performing fluorescent quantitative PCR detection on the extracted genome DNA, wherein the fluorescent quantitative PCR detection is represented by '1' when a melting curve peak is formed, and the fluorescent quantitative PCR detection is represented by '0' when no melting curve peak is formed; and obtaining a typing result for binary data consisting of 0 and 1. The invention mainly monitors the fluorescence change of a sample to be detected in real time based on the fluorescence quantitative PCR so as to complete the detection of the sample. The invention does not need the step of electrophoretic analysis, which is complicated in operation, thereby having the advantages of simple and convenient operation and high automation degree.

Furthermore, the real-time fluorescence PCR-based vibrio parahaemolyticus typing method provided by the invention selects 18 gene loci of the LVPC gene cluster, designs primers aiming at the 18 gene loci, performs fluorescence quantitative PCR amplification to obtain binary data, and forms a typing fingerprint map, so that the method provided by the invention has a good typing effect.

Drawings

FIG. 1 is a graph showing the melting curves of the genes in tube 1 in example 1;

FIG. 2 is a melting curve of the VP0383 locus in example 1;

FIG. 3 is a melting curve of the VP1091 locus in example 1;

FIG. 4 is a melting curve of the VP1778 locus in example 1;

FIG. 5 is a melting curve of the VP2902 locus in example 1;

FIG. 6 is a melting curve of the VPA0074 locus in example 1;

FIG. 7 is a melting curve diagram of the VPA0716 locus in example 1;

FIG. 8 is a graph showing the melting curves of the respective genes in tube 2 in example 1;

FIG. 9 is a melting curve of the VP0635 locus in example 1;

FIG. 10 is a melting curve diagram of the VP1393 locus in example 1;

FIG. 11 is a melting curve of the VP1563 locus in example 1;

FIG. 12 is a melting profile of the VPA0895 locus in example 1;

FIG. 13 is a melting profile of the VPA1336 locus in example 1;

FIG. 14 is a melting curve of the VPA1708 locus in example 1;

FIG. 15 is a graph showing the melting curves of the respective genes in tube 3 in example 1;

FIG. 16 is a graph showing the melting curve of the locus of VP2132 gene in example 1;

FIG. 17 is a melting plot of the VPA0440 locus in example 1;

FIG. 18 is a melting curve of the VPA1199 locus in example 1;

FIG. 19 is a melting curve of the VP1351 locus in example 1;

FIG. 20 is a melting curve of the VPA1256 locus in example 1;

FIG. 21 is a graph showing the melting profile of the RPI08 locus in example 1.

Detailed Description

The invention provides a method for typing vibrio parahaemolyticus based on real-time fluorescent PCR, which comprises the following steps: extracting the genome DNA of vibrio parahaemolyticus, and performing fluorescent quantitative PCR detection on the extracted genome DNA, wherein the fluorescent quantitative PCR detection is represented by '1' when a melting curve peak is formed, and the fluorescent quantitative PCR detection is represented by '0' when no melting curve peak is formed; and obtaining a typing result for binary data consisting of 0 and 1.

According to the invention, before extracting the genome DNA of the vibrio parahaemolyticus, the vibrio parahaemolyticus with different sources is preferably collected. The invention preferably performs purification culture on the collected vibrio parahaemolyticus. The method of the present invention for the purification culture is not particularly limited, and a method of the purification culture known to those skilled in the art may be used.

In the present invention, the method for extracting the genomic DNA of Vibrio parahaemolyticus is not particularly limited, and a method for extracting the genomic DNA of a microorganism known to those skilled in the art, for example, CTAB method or kit method, may be used.

In the invention, the gene detected by the fluorescent quantitative PCR is preferably an LVPC gene cluster; the gene sites of the LVPC gene cluster preferably include VP0383, VP1091, VP1778, VP2902, VPA0074, VPA0716, VP0635, VP1393, VP1563, VPA0895, VPA1336, VPA1708, VP1351, VP2132, VPA0440, VPA1199, VPA1256 and RPI 08.

In the invention, the VP0383 has a nucleotide sequence shown in a sequence table SEQ ID No. 1. In the invention, the amplified forward primer of VP0383 has a nucleotide sequence shown in a sequence table SEQ ID No. 19; the amplified reverse primer of VP0383 has a nucleotide sequence shown in SEQ ID No.20 of the sequence table.

In the invention, the VP1091 has a nucleotide sequence shown as a sequence table SEQ ID No. 2. The amplified forward primer of the VP1091 has a nucleotide sequence shown in a sequence table SEQ ID No. 21; the amplified reverse primer of the VP1091 has a nucleotide sequence shown in a sequence table SEQ ID No. 22.

In the invention, the VP1778 has a nucleotide sequence shown in a sequence table SEQ ID No. 3. The amplified forward primer of VP1778 has a nucleotide sequence shown in SEQ ID No.23 of the sequence table; the reverse primer amplified by the VP1778 has a nucleotide sequence shown in a sequence table SEQ ID No. 24.

In the invention, the VP2902 has a nucleotide sequence shown as a sequence table SEQ ID No. 4. The amplified forward primer of VP2902 has a nucleotide sequence shown in a sequence table SEQ ID No. 25; the reverse primer amplified by the VP2902 has a nucleotide sequence shown in a sequence table SEQ ID No. 26.

In the invention, the VPA0074 has a nucleotide sequence shown as a sequence table SEQ ID No. 5. The forward primer amplified by the VPA0074 has a nucleotide sequence shown in a sequence table SEQ ID No. 27; the reverse primer amplified by the VPA0074 has a nucleotide sequence shown in a sequence table SEQ ID No. 28.

In the invention, the VPA0716 has a nucleotide sequence shown in SEQ ID No.6 of a sequence table. The forward primer amplified by the VPA0716 has a nucleotide sequence shown in a sequence table SEQ ID No. 29; the reverse primer amplified by the VPA0716 has a nucleotide sequence shown in a sequence table SEQ ID No. 30.

In the invention, the VP0635 has a nucleotide sequence shown in SEQ ID No.7 of the sequence table. The amplified forward primer of the VP0635 has a nucleotide sequence shown in a sequence table SEQ ID No. 31; the amplified reverse primer of VP0635 has the nucleotide sequence shown in SEQ ID No.32 of the sequence table.

In the invention, the VP1393 has a nucleotide sequence shown in a sequence table SEQ ID No. 8. The amplified forward primer of the VP1393 has a nucleotide sequence shown in a sequence table SEQ ID No. 33; the amplified reverse primer of the VP1393 has a nucleotide sequence shown in a sequence table SEQ ID No. 34.

In the invention, the VP1563 has a nucleotide sequence shown in a sequence table SEQ ID No. 9. The amplified forward primer of the VP1563 has a nucleotide sequence shown in a sequence table SEQ ID No. 35; the reverse primer amplified by the VP1563 has a nucleotide sequence shown in a sequence table SEQ ID No. 36.

In the invention, the VPA0895 has a nucleotide sequence shown in a sequence table SEQ ID No. 10. The forward primer amplified by the VPA0895 has a nucleotide sequence shown in a sequence table SEQ ID No. 37; the reverse primer amplified by the VPA0895 has a nucleotide sequence shown in a sequence table SEQ ID No. 38.

In the invention, the VPA1336 has a nucleotide sequence shown as a sequence table SEQ ID No. 11. The forward primer for amplification of the VPA1336 has a nucleotide sequence shown in a sequence table SEQ ID No. 39; the reverse primer for amplification of the VP1336 has a nucleotide sequence shown in a sequence table SEQ ID No. 40.

In the invention, the VPA1708 has a nucleotide sequence shown in a sequence table SEQ ID No. 12. The forward primer amplified by the VPA1708 has a nucleotide sequence shown in a sequence table SEQ ID No. 41; the reverse primer amplified by the VPA1708 has a nucleotide sequence shown in a sequence table SEQ ID No. 42.

In the invention, the VP1351 has a nucleotide sequence shown in a sequence table SEQ ID No. 13. The amplified forward primer of the VP1351 has a nucleotide sequence shown in a sequence table SEQ ID No. 43; the reverse primer amplified by the VP1351 has a nucleotide sequence shown in a sequence table SEQ ID No. 44.

In the invention, the VP2132 has a nucleotide sequence shown in a sequence table SEQ ID No. 14. The amplified forward primer of the VP2132 has a nucleotide sequence shown in a sequence table SEQ ID No. 45; the reverse primer amplified by the VP2132 has a nucleotide sequence shown in a sequence table SEQ ID No. 46.

In the invention, the VPA0440 has a nucleotide sequence shown in a sequence table SEQ ID No. 15; the forward primer amplified by the VPA0440 has a nucleotide sequence shown in a sequence table SEQ ID No. 47; the reverse primer amplified by the VPA0440 has a nucleotide sequence shown in a sequence table SEQ ID No. 48.

In the invention, the VPA1199 has a nucleotide sequence shown in a sequence table SEQ ID No. 16. The forward primer for amplification of the VPA1199 has a nucleotide sequence shown in a sequence table SEQ ID No. 49; the reverse primer of the VPA1199 has a sequence table SEQ ID No. 50.

In the invention, the VPA1256 has a nucleotide sequence shown in a sequence table SEQ ID No. 17. The forward primer amplified by the VPA1256 has a nucleotide sequence shown in a sequence table SEQ ID No. 51; the reverse primer amplified by the VPA1256 has a nucleotide sequence shown in a sequence table SEQ ID No. 52.

In the invention, the RPI08 has a nucleotide sequence shown in a sequence table SEQ ID No. 18. The amplified forward primer of the RPI08 has a nucleotide sequence shown in a sequence table SEQ ID No. 53; the amplified reverse primer of the RPI08 has a nucleotide sequence shown in a sequence table SEQ ID No. 54.

In the invention, the Primer is designed by adopting Primer 5.0 software. The method for designing the primer according to the present invention is not particularly limited, and the principle of designing the primer known to those skilled in the art can be used.

In the invention, the LVPC gene cluster gene locus is preferably divided into 3 individual lines to be simultaneously subjected to fluorescent quantitative PCR detection. The grouping of the 3 systems is as follows: the VP0383, the VP1091, the VP1778, the VP2902, the VPA0074 and the VPA0716 are taken as a first system; the VP0635, VP1393, VP1563, VPA0895, VPA1336 and VPA1708 are a second system; the VP1351, VP2132, VPA0440, VPA1199, VPA1256 and RPI08 are in a third system.

In the present invention, the PCR reaction system for the fluorescent quantitative PCR detection preferably includes: 1 mu L of LDNA template, 3 mu L of EvaGreen dye, 10 mu L of PCR premix, 1 mu L of forward and reverse primers respectively and 4 mu L of ultrapure water;

the primers comprise 6 pairs of primers of the same system, and the concentration of the primers is as follows:

a first system: VP 03830.5. mu. mol/L, VP 10910.04. mu. mol/L, VP 17780.12. mu. mol/L, VP 29020.25. mu. mol/L, VPA 00740.26. mu. mol/L, VPA 07160.16. mu. mol/L.

A second system: VP 06350.12. mu. mol/L, VP 13930.6. mu. mol/L, VP 15630.15. mu. mol/L, VPA 08950.18. mu. mol/L, VPA 13360.12. mu. mol/L, VPA 17080.18. mu. mol/L.

A third system; VP 13510.02. mu. mol/L, VP 21320.4. mu. mol/L, VPA 04400.1. mu. mol/L, VPA 11990.1. mu. mol/L, VPA 12560.26. mu. mol/L, RPI 080.4. mu. mol/L.

In the invention, the sources of the Eva Green dye and the PCR premix are not particularly limited, and the Eva Green dye and the PCR premix known to those skilled in the art can be adopted. In the embodiment of the invention, the Eva Green dye is purchased from Biotium; the PCR master mix was purchased from KAPA.

In the present invention, the PCR reaction conditions for the fluorescent quantitative PCR detection are preferably: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 5s, 30 cycles.

In the invention, the high-resolution melting curve is preferably obtained by raising the temperature to 77 ℃ after the last cycle of PCR, preserving the temperature for 5s, and raising the temperature to 93 ℃ at a heating rate of 0.1 ℃/s.

In the present invention, the fluorescence quantitative PCR detection is performed by using a fluorescence quantitative PCR instrument known to those skilled in the art without any particular limitation. In the present example, Bio-Rad CFX 96 was used^TMThe above process is carried out.

After the fluorescent quantitative PCR is finished, the obtained melting curve is called out of a system, typing analysis is carried out according to the difference of melting curve fingerprints of all the strains, the melting curve fingerprints are the same type if the melting curve fingerprints are the same, and the melting curve fingerprints are different types if the melting curve fingerprints are different. Specifically, the melting curve peak of the fluorescent quantitative PCR detection is represented by '1', the melting curve peak of the fluorescent quantitative PCR detection is represented by '0', and 18 gene sites of the LVPC gene cluster of each vibrio parahaemolyticus form binary data consisting of '0' and '1'.

The method for the typing of Vibrio parahaemolyticus by real-time fluorescent PCR according to the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

53 strains of vibrio parahaemolyticus are stored in the laboratory, and the vibrio parahaemolyticus strains are separated from food or diarrhea patients. The isolation and identification of 53 strains of Vibrio parahaemolyticus were carried out strictly according to the national standard GB 4789.7. The genomic DNA of 53 strains of Vibrio parahaemolyticus was extracted using KAPA DNA extraction kit according to the protocol.

The extracted genome DNA is configured into a PCR reaction system, specifically 1 mu L of LDNA template, 3 mu L of Eva Green dye, 10 mu L of PCR premix, 1 mu L of forward and reverse primers respectively and 4 mu L of ultrapure water.

The primers comprise 6 pairs of primers of the same system, and the concentration of the primers is as follows:

a first system: VP 03830.5. mu. mol/L, VP 10910.04. mu. mol/L, VP 17780.12. mu. mol/L, VP 29020.25. mu. mol/L, VPA 00740.26. mu. mol/L, VPA 07160.16. mu. mol/L.

A second system: VP 06350.12. mu. mol/L, VP 13930.6. mu. mol/L, VP 15630.15. mu. mol/L, VPA 08950.18. mu. mol/L, VPA 13360.12. mu. mol/L, VPA 17080.18. mu. mol/L.

A third system; VP 13510.02. mu. mol/L, VP 21320.4. mu. mol/L, VPA 04400.1. mu. mol/L, VPA 11990.1. mu. mol/L, VPA 12560.26. mu. mol/L, RPI 080.4. mu. mol/L. The whole detection system is divided into 3 tube reaction systems, and each 6 gene primer pairs are 1 tube reaction system. The primer grouping information is detailed in table 1.

TABLE 1 primer List

The reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 5s, and fluorescence collection at the extension stage. And finally, monitoring a melting curve, namely after the last cycle is completed, raising the temperature to 77 ℃, keeping the temperature for 5s, gradually raising the temperature to 93 ℃ at the temperature rise rate of 0.1 ℃/s, and continuously detecting the fluorescence intensity in the temperature rise process. All reactions were performed in a fluorescent quantitative PCR instrument Bio-Rad CFX 96^TMThe above process is carried out. The formed high-resolution melting curves of each gene locus are shown in the attached figures 2-21.

Binary data are set for 18 gene sites of the LVPC gene cluster of each Vibrio parahaemolyticus strain according to the existence of a melting curve peak, and are represented by '1' when the melting curve peak is detected at the site by fluorescent quantitative PCR, and are represented by '0' when the melting curve peak is not detected at the site by fluorescent quantitative PCR, the binary data consisting of '0' and '1' are formed for 18 gene sites of the LVPC gene cluster of each Vibrio parahaemolyticus strain, and the binary data of 53 Vibrio parahaemolyticus strains are shown in Table 2.

Table 253 Binary data List of Vibrio parahaemolyticus strains

The melting curves of 18 loci are shown in FIGS. 2-7, 9-14 and 16-21, and it can be seen from the melting curves that the temperature corresponding to the melting peak value reached by amplification of the VP0383 primer pair is 81.8 +/-0.5 ℃; the corresponding temperature of the VP1091 primer pair when the amplification reaches the melting peak is 89.4 +/-0.1 ℃; the corresponding temperature of the VP1778 primer pair when the amplification reaches the melting peak is 87.1 +/-0.3 ℃; the corresponding temperature of the VP2902 primer pair when the amplification reaches the melting peak is 84.1 +/-0.3 ℃; the corresponding temperature of the VPA0074 primer pair when the amplification reaches the melting peak value is 88.5 +/-0.3 ℃; the corresponding temperature of the VPA0716 primer pair when amplification reaches a melting peak is 85.6 +/-0.4 ℃; the corresponding temperature of the VP0635 primer pair when the amplification reaches the melting peak is 87.4 +/-0.4 ℃; the corresponding temperature of the VP1393 primer pair when the amplification reaches the melting peak is 85.5 +/-0.5 ℃; the corresponding temperature of the VP1563 primer pair when the amplification reaches the melting peak is 84.1 +/-0.6 ℃; the corresponding temperature of the VPA0895 primer pair when the amplification reaches the melting peak is 90.5 +/-0.1 ℃; the corresponding temperature of the VPA1336 primer pair when the amplification reaches a melting peak value is 81.3 +/-0.4 ℃; the corresponding temperature of the VPA1708 primer pair when amplification reaches a melting peak is 89.5 +/-0.2 ℃; the corresponding temperature of the VP1351 primer pair when the amplification reaches the melting peak is 89.3 +/-0.2 ℃; the corresponding temperature of the VP2132 primer pair when the amplification reaches the melting peak is 81.6 +/-0.6 ℃; the corresponding temperature of the VPA0440 primer pair when the amplification reaches the melting peak is 86.9 +/-0.4 ℃; the corresponding temperature of the VPA1199 primer pair when the amplification reaches the melting peak is 88.3 +/-0.4 ℃; the corresponding temperature of the VPA1256 primer pair when the amplification reaches the melting peak is 83.3 +/-0.5 ℃; the corresponding temperature when amplification of the RPI08 primer pair reaches the melting peak is 85.3 +/-0.6 ℃.

FIG. 1, FIG. 8 and FIG. 15 are the detection analysis maps of a certain strain, the melting curves obtained by each gene as amplification do not overlap each other, and the peak shape is complete, which indicates that the 18 pairs of primers obtained by design have better resolution, and can accurately distinguish the amplification conditions of 18 gene sites.

After the detection of the invention, 53 strains of vibrio parahaemolyticus can be divided into 13 different fingerprint spectrums, and 13 types can be obtained.

From the above examples, the method provided by the present invention uses fluorescence quantification in combination with melting curve technology to analyze LVPCs of vibrio parahaemolyticus, and determines the presence or absence of corresponding LVPCs according to the presence or absence of the melting curve peak. Because the fluorescent quantitative technology is used for analyzing the Vibrio parahaemolyticus LVPCs, the fussy electrophoresis process is saved, and the selected gene locus has better resolution ratio, thereby achieving the purpose of accurate typing. Meanwhile, the method is simple and convenient to operate, and time and labor are saved.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> center for disease prevention and control in Jiaxing city

<120> method for typing Vibrio parahaemolyticus based on real-time fluorescent PCR

<130>2016

<160>54

<170>PatentIn version 3.3

<210>1

<211>540

<212>DNA

<213> Artificial sequence

<400>1

ctaatcgtga tccgtcacaa ttaagtgtgt gtttcttcca actcttttta gatcacgaag 60

tgctgtctct ttcttcgcaa tccgaagggg ccgagctgaa gttatggtat tgttgataat 120

gtcaatatca tcacgtttgg tgtgcgtgaa ctcgataacg ccgtagggag ttctaaactc 180

ccctttacgc cccgttgtca tgatagtaat tctatcggtc attacttgag aaatgacact 240

gtattctgag agtgccgatt ctaaagatat gtagttgtac tctcctcgtc gcaagcattt 300

agcaatgtat tcaatggtat aaccgtcctt atgacgactg tatgagaata cataaacacc 360

gttgcacacc cgctctagta ctccagcttt caccaaacga ggtaaacttt gctggaatgc 420

tgtaagtgag tcatcaaaaa ataatctcct gaggtcactt tttcggtaaa cgtgtatacc 480

tcgcctgtca aatttatgca ggcgatccaa tgcatcccca atcttcactt caatgctcat 540

<210>2

<211>1140

<212>DNA

<213> Artificial sequence

<400>2

atgagacgta gaaccacgct atcgctgctg attgcatcga gcctgtttat ggcgggatgt 60

caaccaagcc aagaaggaac tgagcaaggt ggtggtggcg ctcccgcaac cgaagtgaat 120

gtgttgacgg ttgaacctgt tcgacaagct ttgacggttg agttgccggg ccgcagccgt 180

gcatttaaag aggcggaagt ccgcccacag gtaacgggta ttatttcaga acgtaacttt 240

gttgaaggtg gcgttgtgga aaaaggtcag tcgttgtatc aaattgatga ctcttcattt 300

caagcggatt tgcttagtgc agaagcggag ctgattcgtg cgcaagcttc tgaagaatcg 360

acgtttgcaa ccgttaaacg ttaccgcgca ttgattacca agaaatcgat cagtcaacaa 420

gatctggatg aagcagaagc ggcttacaaa gaagcgaaag cgcaagtgct cgtagccaaa 480

gcgaagatca atactgcaaa gatcaatttg acttataccc gtgtgaaagc gccgatcagt 540

ggcgtgatca gcaagtcaaa tgtgactgcg ggagcgttgg ttaccgcaaa ccaagccgac 600

aagctaacga cgattcaaca acttgatccg atcaacgtgg atatcgttca gtcttctgct 660

caattgctgc gactaaaagc ggcgctttct caaggtcaca tgcaagaaga tcaatctgcg 720

caagtcgcat tgacgcttga agatggctcg acttacgagc acaaaggcac aatgaagttc 780

acggaagtta acgtggatga aagtacggga agcgtgactt tgcgagcgga attcccaaat 840

cctgatggtt tacttcttcc gggtatgttc gtgcgtgcga ccgtgattac tggtgttgac 900

cctagcgcga ttttgatccc gcaaaacacc gtgactcgcg atgcaacagg caaagcttcg 960

gtgatgacag tgagtgctga taataccgtt gcgattacgc cagtcatcac ggcggaagtt 1020

atagacaacc aatggcgaat cattgatgga ttaaaagccg gtgatcaagt cattacagcg 1080

ggtttgcaaa aggtgcgtcc gggtagccca gtcaccatcc aaacgggaga gcagggataa 1140

<210>3

<211>558

<212>DNA

<213> Artificial sequence

<400>3

ctaaaatgtg gttggcgtat gcgcacttat caatcgacaa gccttgtctg tgtgattcgt 60

aaatcgatgt ggcttcgtcg tatcaataat gtacgactcg ccggctgtga ggacgtagga 120

gtggcctttg tactctagag tgatttctcc ctctaaaatc gtgccgacct cttcgccttc 180

gtgtctaatt tccgccgcac ctgttgttcc atgtggtgcg tattcttcga tcaagaatcc 240

aataacttga tctttgtcgc cattcgtcac tagtttcatg gataccgtct cgctcccaat 300

ttccacgaga tctgacggtt tgaagaccac tttcacttca tcattcgaag attgttcaaa 360

aatgaaaaac tccgacagtg ataaagaaaa tacattgacg attttttgta atgagctgac 420

cgacgggctc actttgccat tttcaatgga cgagattgca ctgtgggtaa taccagcgcg 480

ttcggctaat tccctttgtg acaatccatg ttttttacgt aactgaacaa tatttttacc 540

aatctcatga ttgtccat 558

<210>4

<211>1134

<212>DNA

<213> Artificial sequence

<400>4

atgcacactt ttggaaaagt tcagcaacta acagcaaaca taaaatcagt tgatttgttt 60

aacttattct cattatttga cgtcccctca gaactcgaag aaagctttca agcaatacat 120

agtcgcagga ataagagacg aatcgagtta gcgaagagcg cccttgagtc aggcttagac 180

aattctattg aagttccaca accatctctt acttttgttg taggggaagt tttaagccac 240

aaaaacttag gtagaggctt aatagagata gagtatgacc ctttagacac tttgattgtg 300

gatggcgtaa tcactttgtt tgccataatg cagctaagcg gctttagtca tccatttgag 360

aaaaaacgag tctcgaaaga gttaatccta aaaaatgacg ttgctagaca agagcttgca 420

cattgtccta tacaggtgaa cttgttgttc tcaccaacag agcctttatc aaaaaaaact 480

tgtattaccc tttacaagaa atacagccaa actgaaaaaa atattcatgc tcccttaatt 540

gaaagtgtaa atgcggagtt accaattaat acttatgtaa gagaggtcgc gaaaacaata 600

gatcttcaag cttttggcgg catgaataca acatcaatta gactctcagt taaagaccct 660

tatgtgacga ctgaggcaac gatgattcga ttagttctcg gggcaattgg tggggcggat 720

taccaagata aaaataaagt tgatgtgttt ggttcagggc cgtttagtgc aaaacatact 780

aacgtaataa agccctatat ctgtatattt atggaagcat ggttgaaatc tgttaaggga 840

caactattct cacacaagag tggttttcat tactcaacga cgctttggca gtcactaggg 900

ctagtgatac ataagctttt tcttaacgag gagcctatca atgagtttgc caaagcagga 960

gcatttttag gtcaattaga ttactccaaa tcagctaaac actgggggga ttgcgatgct 1020

cttgaactcg atgcttccgg gaagctctac aaaaacgcca caggtggagg acgagctatt 1080

cgtattgcaa tggctcacta cctattcaac gtttatcaaa acgggaaaaa gtaa 1134

<210>5

<211>711

<212>DNA

<213> Artificial sequence

<400>5

tcagcgcgtc aactgacgca acgtgacatc cggaagcgcg gtcagttttt tactttgtac 60

taagaacaat tccgcgcacg ctaaggcatc actggcggct gagtgcgata agtaccccgg 120

caggtgatag tagcggcgca aatcgtccaa ctgataactg ctgtgcgcgc cgcttttccc 180

tgcatagcta aactgctttt cgatttgcag agtgtcgata aaatacgcgg ggaaatcctc 240

tacttggtag cgagaggaaa agtaagcgtg aatgaaggct ttttcaatgc agcatccgtg 300

ggcgagcact acttttcctg tgatgcgttt cagcaaccga ttcatcccct catccaatgg 360

cagtccttta gcgagatctt gcggagttaa gccattaatt ttcgctgttt ctggtttgat 420

gaattgtccg tgacaaatgt accactcttt ggagctggcg atatcaatgc cgtccagcgt 480

taggtcaacc acgccaatcg ataagatttt atcaccttgc gcgtccacgc ctgtggtttc 540

aaagtccaac gcaacaaaac tcaaatcctt caaggtcgtt gagtcatcca ccaaaggccg 600

ttgaagatag tctttcagca ccattggcca atcgtcaaca acgtgaatgg cttggcgttt 660

ttgctcagga gagagcggcg tttcccaccg cttaaaccaa cgtttaatca t 711

<210>6

<211>984

<212>DNA

<213> Artificial sequence

<400>6

ttgaaggaaa ccaataagaa aaacctccgt gtcgtagcgc ttgctccgac agggaggtat 60

tttgcatcaa taataagctc attagagatc ttggaaacag cagctgaatt tgcagagttc 120

caaggtttca tgacacacgt cgttacgcca aacaatcggc cgttaattgg tcgagggggg 180

atcagcgttc aacccaccgc acaatggcaa tcatttgatt ttacaaacat attaatcatt 240

ggctcaattg gcgatccttt agaatcgtta gataatatcg atcctgcatt gttcgattgg 300

atcagagagt tacacctaaa aggcagcaaa atagttgcaa ttgacactgg tatttttgtc 360

gtcgcgaaag ccggtctttt gcagcaaaac aaagctgtaa tgcattctta cttcgcccac 420

ctctttggcg aactgttccc tgagataatg ctcatgactg agcagaaagc actcattgac 480

ggaaacgtct atctttcgtc tggaccttac tctcactcaa gtgtgatgct ggaaatagta 540

gaagagtatt ttggaaagca cactcgaaac cttggtaacc agtttttaag tacaatagag 600

agcagtggaa actctcattc atactgtgat gttttccgtt acatgcaaca cagagatgag 660

ctgattctaa aaatccaaaa atggattctc accacggatc tagacatcgt ctctatcagt 720

gatttagcga atgaagcttg cttatcagaa cggcagttaa aacgccgatt taaagaggcg 780

acatccatca gtccgctaaa gtttattcag ttagggcgtt tatcttttgc aaaagaactg 840

ttgaggtcaa caaagttgtc aattgatgaa gttgcaagtc gatctggtta tgttgataca 900

cagtttttca ggcagatatt caaacgagaa aatgattgtt ctccgcttga gtatcgtaaa 960

agaaaccaag ttaaagctga atga 984

<210>7

<211>891

<212>DNA

<213> Artificial sequence

<400>7

ttaggtatga tgggtaagtt tgtcttttaa ccagtcatga aaaagtgcca cttttcttga 60

agttaaccgg aagtttggga tcaccacata atagccatag ccgctgtgaa gctttagatt 120

cccaatatgt tggatgtcac cacgtaaaat agaaaattgc gccataaaat cgggcagcaa 180

agccaagccc ttctccattc tcacggcttc acacgccaaa taaaaatgct caaacccgac 240

ggagtgatag gtgattgggc actctaaatc gttctcctgt ttgaactgct cccacagctg 300

cgggcgagtc gtttgcggaa taaatgggta atggctgata gcttggttgt ctgacagttt 360

tggtaggttg gtgttgccaa tcagcaacaa ggtttcctca cataagagct ggctatattc 420

ataatgagta gaaagaggca aacaccgcac atgcagatca ctttcgccat gaatgttttt 480

aactgcgcca tctccggtaa gaattttcac gcgaatattt ggatggcgct ggtggaaatc 540

attgatattt ggcacgagcc aaagactggc aaatgagggt gtgacatcga caaccaacag 600

ctcttgatcg gtattggtct gaattaggct ggcggttgcg tgctgaatga gctccagagc 660

ttctgttatt tgaggcaaat agcgctttcc ttcctctgtt agctccacgc cgtttaaccc 720

tcgatgaatg agaggaagcc caacgagttg ctctaaagat gccatctgtt tactgaccgc 780

tccttgtgtt acgcacaggt catcggcggc tttagaaaag ctgagatagt tagccacagc 840

aacaaaagtc ctcaacgcac gaatggaagg gtactcactg ccttttatca t 891

<210>8

<211>519

<212>DNA

<213> Artificial sequence

<400>8

atgccaactc cagcatatat gtctatcaac ggtgaaactc aaggtcacat tactaaagat 60

acatactctg cagattcagt aggtaacaca tggcaagaag ctcacgttga tgagttccta 120

gtacaagaac ttgatcacgt gctaactgtt ccacgtgatc cacaaagtgg tcaacctact 180

ggtcaacgtg tacaccgtcc acttgttgtg actaaagttc aagaccgttc ttctccacta 240

ctatttaacg cactagtgtc tggtgaaaaa cttcctgagt gtttgattcg tttctaccgt 300

acttcagttc aaggcaagca agagcactac tactcaatca agctgattga tgcgctattg 360

gtagatatcc aaactcgtat gaaccactgt caagatgcag caacagctga tcgcgttaca 420

gaagaagttc ttaagtttac ttaccgtgca atcgaagtaa ctcacgaaaa ctgtggtaca 480

gctggtaacg acgactggcg tgctccacgcgaagcataa 519

<210>9

<211>429

<212>DNA

<213> Artificial sequence

<400>9

atgtttcacg aatcattccg cacactcttt tggcgtgagt ttacctccat taagcaaggc 60

gctgaatatt ttcacgtatc caaacccacg attactcgtt ggcttgatgg tacggtttct 120

atcaatccaa tggcagaaaa actactgttg attaaggcgc ttggttattt gcctaatgat 180

ttgcgttggt ctgggtttcg tatctgtgaa aaacgagctg tgtttatcac accgtctggt 240

cgtgagttta gccctaaaga attggaaagc tttgtgttct ggcgtgacga acatcgtcag 300

tttgtggaaa tgtacggaca ctttgagtat cccaaggttt atcccgctaa agaaaacgtc 360

ttaccgtttc gtggcggccg tcgaatgaaa gccgccaagt ggataccaag taagaagcga 420

attacctag 429

<210>10

<211>717

<212>DNA

<213> Artificial sequence

<400>10

ttaaatctcc ataatcgtga cggtgtcacc cgccgtgcct gttaatgtca tggcggccgt 60

gctttcgatg gtgatgcgct cgcccgcgtc cagttcaaac gcgtcaacaa acaccgaggc 120

agtgttggct ttcgacgcct taatcgtgat ggctttgcgc gtggtgtttt gtgccatgtt 180

gtgcggaaac acgctcacgg cttcggtgat gaggttgcta ctgacttgct catccacttt 240

gaccacttgc gtcgcttcga gcgtgaccgc gggcaacgat tgaacattca cgggttgcgg 300

cgctttgaac tcaacaggcg gcaaagtggt gacgccgagc tgttgattcg cggccaattc 360

aaccgcggga agctgattca ctgccaattg ttgattgggc gcgatttcta ccgcgggtaa 420

ctgttcaatc accacactcg gcaaggcttg aaccaccacg ctttgaccct caacaggcgg 480

cataaatgat ccataaccaa actgaatttc gatttcgttg tcggtgcggc tagagataag 540

caaacgccca aggtgtttac cctcgcctac gttaaacacc gccgacttgc caagcgtgac 600

gcgctcaccg gacgattcac gatagatttc aatttcagct tgcgccgctt tcagatacag 660

ccaattgcca tcaggcgtca gcgggatggc ttgacccgcg ataagttgcg tgttcat 717

<210>11

<211>753

<212>DNA

<213> Artificial sequence

<400>11

tcaataaccg tcattataac ctcgcataaa attaactaat tttccatttt caaattcaaa 60

tatataatat tttttcttat ataacactcg gtacttattc aaggtattga tttgattaaa 120

ataatttaca gaaagatcaa aactcatcgt actagctagg gggatagcta tacttcttgt 180

atagttctct acgtttatat attttttaaa atcactactc aaatacgcga attcattatt 240

ttttgaatat attttttcat ctattatgct aacaaaatta ttattatcaa agtgaagaga 300

attgaatact ctaagtccag gagtattttt ttctaatcta actttcaccc cctctatctc 360

aatctttctc cacttaaaat tataattcga ttgctgatac tgcagcgtca gaacaatctc 420

gttttgctca atatcgcgtt ttttattggt aggtctgata tagacatttt tcgtaggatg 480

acggaataaa aaatttaata aacccctttc tttaaccgat tcaatacttc gtgtattctg 540

caataattct gtatcacact cctcatcctc atcctcttct tcctgatcaa gtacaaagct 600

aagatcaatg tctctgctct cgactttact catttgattc agaattttag agaagtcatc 660

atcagtatta gattgtttag aggttgcttt atcaaattca ccgtctaccg tatctcgaat 720

gaatagcgat acagaattat taatgttgct cat 753

<210>12

<211>630

<212>DNA

<213> Artificial sequence

<400>12

atgtctagca ttaaagaaca attgaaagct ctgaaagtca tccctgttat cgccatcgat 60

aaagcagaag acattatccc actgggtaaa gtactggcag aaaacggact cccagccgct 120

gaaatcacct tccgttcagc agcggctgca gaagcaattc gcctactgcg tgaaacccag 180

ccagacatgc tgattggcgc aggtacagta ctgaatcgtg agcaagcgat cgcagcaaaa 240

gaagcaggcg caacctttat cgtatcgcct ggctttaacc caaataccgt caaagcgtgc 300

caagaaatcg gcattgatat cgtaccgggc gtgaacaacc caagcacggt tgaagccgca 360

ctggaaatgg gattaaccac cttgaagttc ttccctgccg aagcttctgg cggcatcaac 420

atggtgaaat cactgctcgc gccctacacc gacatcgaac tgatgccaac aggtggcatt 480

aaccctgcga acatcaaaga ttacttagcg attccacgcg tgttggcctg cggcggaact 540

tggatggtag acaagaaact gatcgaagaa ggtaactggg aagagcttgc acgcctaact 600

cgcgaagcgg ttgcgcttgt aaatgagtaa 630

<210>13

<211>1038

<212>DNA

<213> Artificial sequence

<400>13

atgaaattag caaccaagaa aaatgggact cgtgacggcc tgttgatggt cgtgagcaaa 60

gacttaacgc gctgcgtacc cgcaacagaa gtgttccacc cgagaaacct agcaccgact 120

atgcaagtcg cgctcgataa ttgggaagct gttgcaccac aattggaaga aatttacacc 180

gcgctgaaca acggcacggt cgcagggttt gaagagtttg aggcgcatta ctgtgagtct 240

ccgttacctc gcgcttatca gtgggcggat ggcagtgctt atgtcaacca tgtcgagttg 300

gttcgtaaag cgcgcggcgc agaaatgcca gagagttttt ggactgatcc tctgatgtat 360

caaggtggtt cagatgcgtt tattggccct tgcgacaata ttgagtttgc cagcgacgaa 420

tggggtatcg attttgaagg cgaagttgcc gttgtaactg gagatgttcc gatgggcgca 480

agtattgctg aggcgcaaga gtctattcgt ctcattatgt tggtgaacga cgtctcgctg 540

cgtggtttga ttcctgcgga gctcgcaaaa gggtttggtt tcttccaatc taaaccgtct 600

tcggcgtttt ctcctgtagc ggttacccct gatgaattgg gcgatgaatg gtatgaaaac 660

aaagttcatt taccattggt ttccacctac aaccacaagc cgtttggtcg ccctaacgct 720

ggcgttgata tgacttttga ctttgcagat ttaatcgttc atgccacaaa aacgcgccca 780

ctatcggctg gcgcaatcat cggttctggc acggtgtcca acaagcaagg cactgaccat 840

ggcacttcga ttgaggaagg tggcgttggt tattcatgta tcgcggaagt ccgtatgatc 900

gaaaccattc gtgatggcaa gccaacgaca aactttatgt cgtttggtga ttcgatcaaa 960

cttgagatgt ttgatgttga aggtaatact atttttggtg ctattgatca gcaagttagt 1020

caatacttga aacattaa 1038

<210>14

<211>384

<212>DNA

<213> Artificial sequence

<400>14

atgcgtaaac tagcaaattt tgagcttaaa gcgattctag aatccattga acatgctgat 60

tttacgaata attatgactt cttcaatctg ttaagttgtg ggatatcaat tgaacagatt 120

agagcagtat atagactcag atcagctttg cagcgtgact cttctaaaac aatcttcgat 180

tatccctcgt ttcttgctca aatttactcg aatgttgact ccttctactc tgtcaagcag 240

gctcttggat acgaggttga tacccaagaa atagagcggg aagataagct tatatttctt 300

atgcagtttt tagctgaaaa caaaatgcaa ctcagtatag accgccgtaa tacaccgtac 360

tctattgcaa taaatctagg ctag 384

<210>15

<211>510

<212>DNA

<213> Artificial sequence

<400>15

atgaatacac aacttcaaac gacacatcta ttacacaacg ctcagcaccc actaagtatc 60

tattgcgatg gttcagcacc agacaatcag cacgggtgtc ttcaaggggg tgttggtatc 120

gcggtttacg acgctcttgg tcaatgcgtc tatgagtact catcctcggt tcgcaggact 180

ggcggtgtca ccagccaaag ggctgagctt ctggctctca tcattgcctt acttgtagca 240

ggtgaggggg acaatgtctt tagtgacagt cagtactgtg tgcggggctt caatgaatgg 300

cttgatagct ggaagcgtaa cgcttggcgc aactcaagca agaagcctgt cgctaatcag 360

gacttatgga tgctgataga tgagcttaaa gctattcgtc cacgtgttag cgttgagcac 420

gtagctggtc actctggtat caaaggtaat gagcattcag accgcttagc gacccaagca 480

gccgttgatt ctaaacaaac actctcatag 510

<210>16

<211>2490

<212>DNA

<213> Artificial sequence

<400>16

atgaaaatga caagacgtgc gtttgtgaaa gcaaacgcgg ctgcatcagc tgctgctgtc 60

gcaggtatta cactaccagc atctgcagcg aacctgattg caagctctga tcaaaccaaa 120

atcacatggg acaaagcgcc ttgtcgtttt tgtggtacag gctgttctgt tcttgttggt 180

actcaaaacg gcaaagtcgt tgctactcag ggtgacccag aagcaccagt aaacaaaggc 240

ctaaactgta tcaaaggcta cttcctttct aagatcatgt acggtcaaga ccgtctgact 300

caacctcttc ttcgtatgaa agacggcaaa taccacaaag atggcgaatt tacgccagtt 360

tcgtgggacg ttgcattcga tacgatggca gaaaaatgga aagcatctct agagaaaaaa 420

ggcccgacta gcgttggtat gtttggctct ggccaatgga ctgtaatgga aggttacgcg 480

gcagcgaaaa tgatgaaagc cggtttccgt tctaacaaca ttgaccctaa cgctcgtcac 540

tgtatggcgt ctgcggtagt aggtttcatg cgtgctttcg gtatcgatga acctatgggc 600

tgttacgatg acttcgagaa cgctgatgca ttcgtgcttt ggggttcgaa catggcagaa 660

atgcacccag tactatggac tcgtattact gaccgtcgcc tgagccaccc tcatgttcgc 720

gtaaacgtac tttcaactta ctaccaccgt tcattcgagt tggcagatca cggctacatt 780

tttaacccac agtctgacct tgcgattgct aactttatcg cgaactacat catcgaaaac 840

gatgcagtaa actgggattt cgtgaacaag cacacgaact ttactcaagc agacaccgac 900

attggttacg gtctgcgtga cgacgacccg ctacaaaaag cagcgaaaaa ccctaactca 960

ggcaaactga cgtcaatctc tttcgaagag tacaagaagt ctgtagcgcc atacacggtc 1020

gagaaagcgt ctgagatctc gggtgttgag aaagaaaaac tgattgagct tgcgaagcaa 1080

tacgccgatc caaacacgaa agtaatgtca ctttggacaa tgggtatgaa ccaacatact 1140

cgcggcgtgt ggatgaacaa cttggtttac aacatccacc ttctaaccgg taaaatcgct 1200

actccgggta atagcccatt ctcactaact ggtcagccat cggcgtgtgg tacagctcgt 1260

gaagttggta cgtttgctca ccgtctacct gcggacatgg tggttgctaa ccctaaacac 1320

cgccaaatcg cagaaaaaat ctggaaactg cctgaaggca cgattccacc aaaacctggt 1380

ttccacgcgg tacttcaaga ccgcatgctg aacgatggcg tactgaactg ttactgggtt 1440

caatgtaaca acaacatgca agcaggtccg aacattaaca ctgaacgtct gcctggctac 1500

cgtaacccag aaaacttcat cgttgtttct gacccatacc caacggctac cgctcaagcg 1560

gctgacctta tccttccaac agcaatgtgg attgaaaaag aaggcgctta cggtaacgca 1620

gaacgtcgta ctcaagcttg gtaccaacaa gtaggcacgg taggcgacgc taagtctgac 1680

ctatggcagg taatggagtt ctctaaacgc ttcaagatgg aagaagtgtg gccagaagaa 1740

cttctagcga aagcacctca gtaccgtggc aaaaccatgt acgacatgct gttcaaaaat 1800

ggccaagttg acaagttccc gcttgaagag gcacgtgaac tgaacgacga ttcgcaccac 1860

ttcggtttct acgttcaaaa aggtctgttt gaagaatacg caacgttcgg ccgcggccat 1920

ggtcacgact tagcaccata cgatgtgtac cacaccgtac gcggtctacg ctggccagtc 1980

gttgatggta aagagacaca atggcgcttc aaagaaggct cggacccata cgcgaaagcg 2040

ggttctggtt gggatttcta cggcaatgcc gacggcaaag cgaagatcat ctctgcaccg 2100

tacgaagcgc cacctgaagt gccagattca gaattcgacc tatggctatg tacaggccgt 2160

gttcttgagc actggcacac tggtaccatg actcgtcgtg tacctgagct gtacaaagca 2220

gtaccggatg cggtgtgtta catgcaccca gaagatgcga aagcgcgcaa cgtacgtcgc 2280

ggtgaagaag tggttatcgc gaacaaacgt ggcgaagtac gcgttcgcgt tgaaacacgc 2340

ggtcgtaacc gtccgccaaa aggcttggta ttcgtaccgt tctttgatgc tcgtattttg 2400

atcaacaaac taatccttga tgcgactgac cctctgtcta aacagacaga ctttaagaag 2460

tgtccagtca aaatcactaa ggttgcttaa 2490

<210>17

<211>1605

<212>DNA

<213> Artificial sequence

<400>17

ctacactagc tctttgacta ttctaagata ctgtaaaaac tctggagctt tcactaagcc 60

ttcgttccaa tactgccgag actttgacat tttatcggca agctcagaag ttaagtattc 120

aaagttttct acaagcctat cgctgccaat aggagagttt ttctctgatg ctagcttctc 180

tgcatatgct tgaatgtgct gacgcatttc aaattttgcg gtttctggtg cttcttcttc 240

gtcggcacaa aagtccaaaa actcacttat ttcggtttca gtctcattac aaagcgcaag 300

tattccctca agatcagcaa tacaacagaa gtggacgttt ttcaaaggta tactagcagc 360

aacagcttca tctattcttt gtctatactc atcccctaaa taagaggcta atttagcacc 420

gttaccaagg tagaagtctt ggtgagtcac tatgagggca tatctgttat caaagtcagc 480

aagatcatca aaaccaacac aacttaactt atctgcacac tcacctgctt gttttatacc 540

tttcagatga gtatccctta acttgtcctt aataatgttg ctctggtcag tcacaagtat 600

tttttggttt ggctcgacgc cctttgcatc aataaatatc gaagttccgt ttcgttgatg 660

taagaagtcg acaaccttac cttgaatttt gttatctcta taaactgcct tcagttgctc 720

ttctcttgtc gtctcaaatc catatgcatc aaaaacatat gccaaatatt cttcaaactc 780

tttagtaaac ttgtctcgaa aacctctcga atcagcagtt ttaagcgttc tgagcacaaa 840

ctcgctcaac ccgatacttg caacgtatgg atgagcggtt gatataccct caggtagaat 900

aagtattggt ttctcaagca atagtggttc agaaaagtac tcttccaccc ttacccctcc 960

atcacctcta gtttctcctc ttcggatttt catcaattgt tgtaactgtt caaagtttgc 1020

tccaactaaa ctgaggtatt gttttattac agtcactgga tatgcaggtg agagcttggg 1080

caagaactct tcgtacttaa tgacaggctt tttaagcgtg taaaatatag aaaagaaaaa 1140

caccgacaaa acaaaaaagt cttccaactc aatatctgtc tgagctttga aactatctcg 1200

aaaagagcga cttgcacctt cattacacat gatcgagaac aacctgacca agaaatagtt 1260

gtgcactatc tggttacttt gaaatctaag ttgtgttaga aaaaacgctc ttagagacaa 1320

ccaaacatta tcctcatcag caaagttggt agcttttccttgtaacgccc aaattttatt 1380

caatatattc caaacatctt tttcagtcgc aggattagcg ccttggttgg gttctagctc 1440

taatgcccac tcaagcatta ggcagacagt ccatggccga cctattgctt taggttcacc 1500

aggataaacg gtatgtagcc gagtcaataa atgttgcacg actgactctt tagtgtagcg 1560

ttttatagag tttcttatct tctgagtatc tgtgctaaat gacat 1605

<210>18

<211>639

<212>DNA

<213> Artificial sequence

<400>18

ttaatccact tttggcagca tacctttgtc tacaatgaaa tcaataattt tttgaagacc 60

aataccctcc ttcaggttgg taaagacgta aggtttttct gggcgcatac gagcagtatc 120

cgcctccata acatccagtg atgcaccaac atacggcgct aggtctatct tattgatcac 180

caacaaatca gagcgggtga tgccagggcc acctttacgt ggtatctttt cgccttcagc 240

aacgtcaatg acataaatgg ttaaatctgc caactctggg ctaaatgttg cgctcaaatt 300

gtcgccacca ctttcgacaa aaaccaaatc taaatttttg tgacgttttg ctaattcttc 360

gaccgccgct aaattcatag aagcgtcctc acggattgcg gtatgaggac agcctcccgt 420

ttcaacacca attatgcgat cagcatctag tgcttgtgcg cgtgttaaaa tttttgcatc 480

ttcttgcgta tagatgtcat tcgtaacgac cgcaatttga tatttgtcac gtattgtttt 540

acataggatt tcaagcaggg ctgttttacc tgaccccact ggacctccga caccaattct 600

cagcggttgt ttgtaatctt gattattata ttcttgcat 639

<210>19

<211>24

<212>DNA

<213> Artificial sequence

<400>19

ctctcctcgt cgcaagcatt tagc 24

<210>20

<211>20

<212>DNA

<213> Artificial sequence

<400>20

gagcgggtgt gcaacggtgt 20

<210>21

<211>20

<212>DNA

<213> Artificial sequence

<400>21

gaaccacgct atcgctgctg 20

<210>22

<211>20

<212>DNA

<213> Artificial sequence

<400>22

cgttacctgt gggcggactt 20

<210>23

<211>22

<212>DNA

<213> Artificial sequence

<400>23

ctaaaatgtg gttggcgtat gc 22

<210>24

<211>21

<212>DNA

<213> Artificial sequence

<400>24

tgacgaatgg cgacaaagat c 21

<210>25

<211>27

<212>DNA

<213> Artificial sequence

<400>25

tcagcaacta acagcaaaca taaaatc 27

<210>26

<211>25

<212>DNA

<213> Artificial sequence

<400>26

gccatccaca atcaaagtgt ctaaa 25

<210>27

<211>20

<212>DNA

<213> Artificial sequence

<400>27

cgcacgctaa ggcatcactg 20

<210>28

<211>21

<212>DNA

<213> Artificial sequence

<400>28

ccaagtagag gatttccccg c 21

<210>29

<211>25

<212>DNA

<213> Artificial sequence

<400>29

aaaccaataa gaaaaacctc cgtgt 25

<210>30

<211>19

<212>DNA

<213> Artificial sequence

<400>30

ctgatccccc ctcgaccaa 19

<210>31

<211>19

<212>DNA

<213> Artificial sequence

<400>31

gccataaaat cgggcagca 19

<210>32

<211>22

<212>DNA

<213> Artificial sequence

<400>32

ggcaacacca acctaccaaa ac 22

<210>33

<211>25

<212>DNA

<213> Artificial sequence

<400>33

gtctatcaac ggtgaaactc aaggt 25

<210>34

<211>25

<212>DNA

<213> Artificial sequence

<400>34

agaaacgaat caaacactca ggaag 25

<210>35

<211>22

<212>DNA

<213> Artificial sequence

<400>35

atcattccgc acactctttt gg 22

<210>36

<211>25

<212>DNA

<213> Artificial sequence

<400>36

agtttttctg ccattggatt gatag 25

<210>37

<211>19

<212>DNA

<213> Artificial sequence

<400>37

gtgtcacccg ccgtgcctg 19

<210>38

<211>18

<212>DNA

<213> Artificial sequence

<400>38

cgccgcaacc cgtgaatg 18

<210>39

<211>26

<212>DNA

<213> Artificial sequence

<400>39

tttctaatct aactttcacc ccctct 26

<210>40

<211>25

<212>DNA

<213> Artificial sequence

<400>40

ccgtcatcct acgaaaaatg tctat 25

<210>41

<211>22

<212>DNA

<213> Artificial sequence

<400>41

gtcatccctg ttatcgccat cg 22

<210>42

<211>18

<212>DNA

<213> Artificial sequence

<400>42

tgcggcttca accgtgct 18

<210>43

<211>25

<212>DNA

<213> Artificial sequence

<400>43

gcaaccaaga aaaatgggac tcgtg 25

<210>44

<211>25

<212>DNA

<213> Artificial sequence

<400>44

cgacatggtt gacataagca ctgcc 25

<210>45

<211>25

<212>DNA

<213> Artificial sequence

<400>45

aggctcttgg atacgaggtt gatac 25

<210>46

<211>23

<212>DNA

<213> Artificial sequence

<400>46

agtacggtgt attacggcgg tct 23

<210>47

<211>29

<212>DNA

<213> Artificial sequence

<400>47

acacaacttc aaacgacaca tctattaca 29

<210>48

<211>19

<212>DNA

<213> Artificial sequence

<400>48

gccagtcctg cgaaccgag 19

<210>49

<211>24

<212>DNA

<213> Artificial sequence

<400>49

tagcgttggt atgtttggct ctgg 24

<210>50

<211>28

<212>DNA

<213> Artificial sequence

<400>50

caactcgaat gaacggtggt agtaagtt 28

<210>51

<211>21

<212>DNA

<213> Artificial sequence

<400>51

tacaagccta tcgctgccaa t 21

<210>52

<211>22

<212>DNA

<213> Artificial sequence

<400>52

cgaagaagaa gcaccagaaa cc 22

<210>53

<211>24

<212>DNA

<213> Artificial sequence

<400>53

tccacttttg gcagcatacc tttg 24

<210>54

<211>19

<212>DNA

<213> Artificial sequence

<400>54

gccctggcat cacccgctc 19

Claims (5)

1. The real-time fluorescence PCR-based vibrio parahemolyticus non-diagnosis purpose typing method comprises the following steps: extracting the genome DNA of vibrio parahaemolyticus, and performing fluorescent quantitative PCR detection on the extracted genome DNA, wherein the fluorescent quantitative PCR detection is represented by '1' when a melting curve peak is formed, and the fluorescent quantitative PCR detection is represented by '0' when no melting curve peak is formed; obtaining a typing result for binary data consisting of '0' and '1';

the gene detected by the fluorescent quantitative PCR is an LVPC gene cluster; the gene sites of the LVPC gene cluster include VP0383, VP1091, VP1778, VP2902, VPA0074, VPA0716, VP0635, VP1393, VP1563, VPA0895, VPA1336, VPA1708, VP1351, VP2132, VPA0440, VPA1199, VPA1256 and RPI 08;

3 individual lines of the LVPC gene cluster gene locus are simultaneously subjected to fluorescent quantitative PCR detection;

the grouping of the 3 systems is as follows: the VP0383, the VP1091, the VP1778, the VP2902, the VPA0074 and the VPA0716 are taken as a first system;

the VP0635, VP1393, VP1563, VPA0895, VPA1336 and VPA1708 are a second system;

the VP1351, VP2132, VPA0440, VPA1199, VPA1256 and RPI08 are a third system;

the nucleotide sequence of the forward primer amplified by VP0383 is shown in sequence table SEQ ID No. 19; the nucleotide sequence of the amplified reverse primer of VP0383 is shown in SEQ ID No.20 of the sequence table;

the nucleotide sequence of the amplified forward primer of VP1091 is shown in a sequence table SEQ ID No. 21; the nucleotide sequence of the amplified reverse primer of VP1091 is shown in the sequence table SEQ ID No. 22;

the nucleotide sequence of the amplified forward primer of VP1778 is shown in sequence table SEQ ID No. 23; the nucleotide sequence of the amplified reverse primer of VP1778 is shown in sequence table SEQ ID No. 24;

the nucleotide sequence of the amplified forward primer of VP2902 is shown in sequence table SEQ ID No. 25; the nucleotide sequence of the amplified reverse primer of VP2902 is shown in the sequence table SEQ ID No. 26;

the nucleotide sequence of the forward primer amplified by the VPA0074 is shown as a sequence table SEQ ID No. 27; the reverse primer amplified by the VPA0074 is shown as a sequence table SEQ ID No. 28;

the nucleotide sequence of the forward primer amplified by VPA0716 is shown in the sequence table SEQ ID No. 29; the nucleotide sequence of the reverse primer amplified by VPA0716 is shown in the sequence table SEQ ID No. 30;

the nucleotide sequence of the amplified forward primer of VP0635 is shown in sequence table SEQ ID No. 31; the nucleotide sequence of the amplified reverse primer of VP0635 is shown in sequence table SEQ ID No. 32;

the nucleotide sequence of the amplified forward primer of VP1393 is shown as SEQ ID No.33 of the sequence table; the nucleotide sequence of the amplified reverse primer of VP1393 is shown as the sequence table SEQ ID No. 34;

the nucleotide sequence of the forward primer amplified by the VP1563 is shown as a sequence table SEQ ID No. 35; the reverse primer of VP1563 has the sequence table SEQ ID No. 36;

the nucleotide sequence of the forward primer amplified by the VPA0895 is shown as a sequence table SEQ ID No. 37; the nucleotide sequence of the reverse primer amplified by the VPA0895 is shown as the sequence table SEQ ID No. 38;

the nucleotide sequence of the forward primer amplified by the VPA1336 is shown as the nucleotide sequence in a sequence table SEQ ID No. 39; the nucleotide sequence of the reverse primer amplified by the VPA1336 is the nucleotide sequence shown in the sequence table SEQ ID No. 40;

the nucleotide sequence of the forward primer amplified by VPA1708 is shown in a sequence table SEQ ID No. 41; the nucleotide sequence of the reverse primer amplified by VPA1708 is shown in a sequence table SEQ ID No. 42;

the nucleotide sequence of the forward primer amplified by the VP1351 is shown in a sequence table SEQ ID No. 43; the nucleotide sequence of the amplified reverse primer of VP1351 is shown in the sequence table SEQ ID No. 44;

the nucleotide sequence of the amplified forward primer of VP2132 is shown in sequence table SEQ ID No. 45; the nucleotide sequence of the amplified reverse primer of VP2132 is shown in SEQ ID No.46 of the sequence table;

the nucleotide sequence of the forward primer amplified by VPA0440 is shown in a sequence table SEQ ID No. 47; the nucleotide sequence of the reverse primer amplified by VPA0440 is shown in a sequence table SEQ ID No. 48;

the nucleotide sequence of the forward primer amplified by the VPA1199 is shown in a sequence table SEQ ID No. 49; the nucleotide sequence of the reverse primer amplified by the VPA1199 is shown as a sequence table SEQ ID No. 50;

the nucleotide sequence of the forward primer amplified by the VPA1256 is shown in a sequence table SEQ ID No. 51; the nucleotide sequence of the reverse primer amplified by the VPA1256 is shown in a sequence table SEQ ID No. 52;

the nucleotide sequence of the amplified forward primer of RPI08 is shown in the sequence table SEQ ID No. 53; the nucleotide sequence of the amplified reverse primer of RPI08 is shown in the sequence table SEQ ID No. 54.

2. The typing method according to claim 1, wherein the nucleotide sequence of VP0383 is represented by SEQ ID No.1 of the sequence Listing;

the nucleotide sequence of the VP1091 is shown as a sequence table SEQ ID No. 2;

the nucleotide sequence of the VP1778 is shown in a sequence table SEQ ID No. 3;

the nucleotide sequence of the VP2902 is shown as a sequence table SEQ ID No. 4;

the nucleotide sequence of the VPA0074 is shown as a sequence table SEQ ID No. 5;

the nucleotide sequence of VPA0716 is shown in SEQ ID No.6 of the sequence table;

the nucleotide sequence of the VP0635 is shown as a sequence table SEQ ID No. 7;

the nucleotide sequence of the VP1393 is shown as the sequence table SEQ ID No. 8;

the nucleotide sequence of the VP1563 is shown as a sequence table SEQ ID No. 9;

the nucleotide sequence of the VPA0895 is shown as a sequence table SEQ ID No. 10;

the nucleotide sequence of the VPA1336 is shown as a sequence table SEQ ID No. 11;

the nucleotide sequence of the VPA1708 is shown in a sequence table SEQ ID No. 12;

the nucleotide sequence of the VP1351 is shown as a sequence table SEQ ID No. 13;

the nucleotide sequence of the VP2132 is shown in a sequence table SEQ ID No. 14;

the nucleotide sequence of VPA0440 is shown as a sequence table SEQ ID No. 15;

the nucleotide sequence of the VPA1199 is shown as a sequence table SEQ ID No. 16;

the nucleotide sequence of the VPA1256 is shown in a sequence table SEQ ID No. 17;

the nucleotide sequence of the RPI08 is shown in a sequence table SEQ ID No. 18.

3. The typing method according to claim 1, wherein the PCR reaction conditions for the quantitative fluorescence PCR assay are: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 5s, 30 cycles.

4. The typing method according to claim 3, wherein the melting curve is measured by raising the temperature to 77 ℃ after the last cycle of PCR and raising the temperature to 93 ℃ at a temperature raising rate of 0.1 ℃/s after 5 seconds of incubation.

5. The typing method according to claim 1, wherein the PCR reaction system for the fluorescent quantitative PCR detection comprises: 1 mu L of DNA template, 3 mu L of Eva Green dye, 10 mu L of PCR premix, 1 mu L of forward and reverse primers respectively and 4 mu L of ultrapure water; the primers comprise 6 pairs of primers of the same system, and the concentration of the primers is as follows:

a first system: VP03830.5 μmol/L, VP10910.04 μmol/L, VP17780.12 μmol/L, VP29020.25 μmol/L, VPA00740.26 μmol/L, VPA07160.16 μmol/L;

a second system: VP 06350.12. mu. mol/L, VP 13930.6. mu. mol/L, VP 15630.15. mu. mol/L, VPA 08950.18. mu. mol/L, VPA 13360.12. mu. mol/L, VPA 17080.18. mu. mol/L;

the third system comprises VP13510.02 μmol/L, VP21320.4 μmol/L, VPA04400.1 μmol/L, VPA11990.1 μmol/L, VPA12560.26 μmol/L and RPI080.4 μmol/L.

Images