CN116004920B - Fluorescence PCR detection method and kit for four different lineages of strains of porcine reproductive and respiratory syndrome - Google Patents
Fluorescence PCR detection method and kit for four different lineages of strains of porcine reproductive and respiratory syndrome Download PDFInfo
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Abstract
The invention relates to the field of molecular biology detection of viruses, and particularly provides a fluorescence PCR detection method and a kit for four strains with different lineages of porcine reproductive and respiratory syndrome. The fluorescence PCR detection method provided by the invention can be used for simultaneously detecting the porcine reproductive and respiratory syndrome virus L1, L3, L5 and L8 series virus strains. The fluorescence PCR detection primer probe provided by the invention can realize simultaneous detection of the porcine reproductive and respiratory syndrome virus L1 system, the L3 system, the L5 system and the L8 system, has no cross amplification reaction with other porcine viruses, has no cross amplification reaction between each partial system, and has stronger specificity; the sensitivity of the primer probe is high, and the lowest detection concentration of the primer probe to the L1 system, the L3 system, the L5 system and the L8 system is 1 copies/. Mu.L; can rapidly identify single or mixed infected clinical samples of L1 series, L3 series, L5 series and L8 series, and has wide application prospect.
Description
Technical Field
The invention relates to the field of molecular biology detection of viruses, and particularly provides a fluorescence PCR detection method and a kit for four strains with different lineages of porcine reproductive and respiratory syndrome.
Background
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a member of the order monoviridae (Nidovirales), arterividae (Arteriviridae), β arterivirus (betaarcerivirus), and the genome is a single positive strand RNA. The infection can cause Porcine Reproductive and Respiratory Syndrome (PRRS), and is mainly characterized by reproductive disorders such as sow abortion, mummy abortion, weaning and the like, and respiratory symptoms of pigs of all ages, thus causing huge economic loss to the global pig industry. PRRSV can be further classified into PRRSV-1 type and PRRSV-2 type, also known as european type and north american type, depending on the genome. In 2010, a method for PRRSV typing based on phylogenetic lineages was proposed. The classification system classifies PRRSV-1 strains into 4 subtypes (subtype I-Global, subtype I-Russian, subtype II and III) and PRRSV-2 strains into 9 lineages (lineages 1-9) based on the phylogenetic relationship of the ORF5 region. Although asia and america have had PRRSV-1 subtype I-reported globally, no other subtype has been reported in areas outside europe, which is predominantly in the form of PRRSV-2 strains.
PRRSV-2 has a high degree of genetic diversity and 9 lineages can be further divided into several sublines. The earliest reported lineage was lineage 5, which occurs in the united states, predominantly in the united states, southern canada, and china. Lineages 8 and 9 were then found throughout the united states. In 2006, our country has seen a prevalence of highly pathogenic PRRSV (HP-PRRSV) characterized by high fever, high infection rate and high mortality, which is characterized by high temperature, high morbidity and high mortality caused by infected pigs, resulting in a huge economic loss to the pig industry. Lineages 3, 4, 6 and 7 are popular in only a few areas: pedigree 3 is prevalent mainly in south China, pedigree 4 is prevalent mainly in japan, and pedigrees 6 and 7 are currently reported only in the united states. In 2013, a similar NADC30 (NADC 30-like) strain with relatively close relativity to a North American NADC30 strain is transmitted into China, and is widely popular in recent years because of the insufficient protection of the current commercial vaccine. At present, the strain of the pedigree 1 is easy to recombine with the strain of the HP-PRRSV and the strain of the pedigree 3 in different modes, and attenuated live vaccines aiming at different strains are overlapped and used in pig farms, so that the strain of the PRRSV popular in China is diversified. PRRSV emergence and prevalence has been recently 30 years, with continued variation leading to new strains, but control of PRRSV has not been substantially progressed worldwide to date, with the effects and hazards remaining. In actual production, diagnosis of strains of different lineages of PRRSV is important for establishment of epidemic prevention and control strategies.
The current methods for detecting the disease mainly comprise traditional virus isolation culture identification, ELISA, common PCR detection and sequence determination. The traditional separation culture identification method has the disadvantages of complicated operation, long time consumption and high diagnosis missing rate; ELISA method is relatively simple and fast, but for trace or impurity samples, the specificity and sensitivity are low, and erroneous judgment can be caused; PCR is a novel molecular biology technology, and since its birth, it has high specificity and sensitivity, and can obtain a large number of target fragments in a short time, making it one of the important means of current invention research. However, the conventional PCR is easy to cause pollution in operation, has higher false positive, and has some limit on detection; compared with the fluorescent quantitative PCR, the fluorescent quantitative PCR has higher specificity and sensitivity, and is widely applied to diagnosis and detection. Therefore, it is necessary to establish a rapid, sensitive and highly accurate PRRSV detection method based on fluorescent quantitative PCR.
Disclosure of Invention
The invention provides a fluorescence PCR detection method and a kit for four strains with different lineages of porcine reproductive and respiratory syndrome, which are used for solving the defect that the existing technology cannot detect and distinguish the strains of porcine reproductive and respiratory syndrome viruses L1, L3, L5 and L8 at the same time.
In a first aspect, the invention provides a kit for distinguishing porcine reproductive and respiratory syndrome virus L1, L3, L5 and L8 strains, wherein the kit comprises four pairs of primers as follows:
primer pair one:
TGTCCAGTCATAATCTGGTGTGGYC(SEQ ID NO.1)，
GTGGRGGYTTATAYGGRACYTGYYT(SEQ ID NO.2)；
primer pair two:
CAAAACTTGCACCCCATTG(SEQ ID NO.4)，
TGTAACCGGCGCCAATG(SEQ ID NO.5)；
primer pair III:
GGCTATATGGTGGGCCCTT(SEQ ID NO.7)，
CGCCCTTAACAAATTTTGTGAG(SEQ ID NO.8)；
primer pair four:
GGCGAMTCAGACAACCRAMC(SEQ ID NO.10)，
ATGAGCCCATATTCTTCYAGR(SEQ ID NO.11)。
each primer pair, y=c or T, r=aor G, m=aor C.
In the kit provided by the invention, the probe matched with the primer pair is as follows: FAM-CRGGTTCYRGRTCCT-MGB.
In the kit provided by the invention, the probes matched with the primer pair II are as follows: VIC-GCCAGACCGACTAGTAGCCA-MGB.
In the kit provided by the invention, the probes matched with the primer pair III are as follows: CY5-CTAGGCACTCCTGGGGTC-MGB.
In the kit provided by the invention, the probes matched with the primer pair IV are as follows: ROX-TTGGRCAARGACTCG-MGB.
When the kit is used for carrying out quadruple fluorescence PCR amplification, the dosages of each primer pair and each matched probe are the same in a fluorescence PC R system.
The invention also provides application of the kit for distinguishing porcine reproductive and respiratory syndrome virus L1, L3, L5 or L8 virus strains under the non-disease diagnosis and treatment purposes.
Specifically, the invention provides a method for distinguishing porcine reproductive and respiratory syndrome virus L1, L3, L5 or L8 series virus strains, which uses primers and probes in the kit, uses porcine reproductive and respiratory syndrome virus as a template to carry out fluorescent PCR amplification, and judges the porcine reproductive and respiratory syndrome virus strains through different probes.
In the method provided by the invention, the usage ratio of the primer pair and the probe to the porcine reproductive and respiratory syndrome virus template in the fluorescent PCR reaction system is (2.5-3): (20-25).
In the method provided by the invention, the fluorescent PCR reaction is performed by the following steps: reverse transcription at 48-51 ℃ for 5-8min, pre-denaturation at 92-98 ℃ for 25-30s, denaturation at 92-98 ℃ for 3-5s, annealing at 58-62 ℃ for 25-30s, and carrying out 35-40 cycles.
The invention has the beneficial effects that:
the fluorescent PCR detection primer provided by the invention can realize simultaneous detection of the porcine reproductive and respiratory syndrome virus L1 system, L3 system, L5 system and L8 system, has no cross amplification reaction with other porcine viruses such as transmissible gastroenteritis virus, porcine foot-and-mouth disease virus, porcine epidemic diarrhea virus, swine fever virus, porcine circovirus type 2, porcine salmonella, porcine pseudorabies virus and the like, has no cross amplification reaction between each partial system, and has stronger specificity; the sensitivity of the primer is high, and the lowest detection concentration of the primer on the L1 system, the L3 system, the L5 system and the L8 system is 1 copies/. Mu.L; can rapidly identify single or mixed infected clinical samples of L1 series, L3 series, L5 series and L8 series, and has wide application prospect.
The invention designs a plurality of pairs of primers and probes for L1 system, L3 system, L5 system and L8 system, and finally determines the fluorescent PCR detection primer probe combination provided by the invention through comparison and screening, so that the simultaneous detection of the L1 system, the L3 system, the L5 system and the L8 system of the porcine reproductive and respiratory syndrome virus can be realized, the detection method has high accuracy, specificity and sensitivity, the detection kit has good repeatability, and tube sealing amplification does not generate aerosol pollution, and the application of the kit can be helpful for controlling the pandemic.
The reason why the quenching fluorophore of the present invention selects MGB is that the TaqMan-MGB probe has the following advantages compared with the conventional TaqMan-TAMRA probe: (1) Increasing the TM value-15 bases on average increases 18 ℃, which can shorten the length of the probe, especially greatly facilitates the design of sequences with high AT content, and increases the TM value difference between paired and unpaired templates. (2) The signal to noise ratio is improved-because the 3' end quenching group of the probe is a fluorescent group which does not emit light, and the position of the fluorescent group is closer to that of the reporter group in space, the experimental result is more accurate, and the resolution ratio is higher.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the specificity detection of the quadruple fluorescence PCR detection kit for porcine reproductive and respiratory syndrome virus L1, L3, L5 and L8 strains and other swine diseases, wherein 1: l1 is CHSX1401 virus; 2: the L3 strain QYYZ virus; 3: the L5 strain VR2332 virus; 4: l8 is JXA1 strain virus; 5: porcine transmissible gastroenteritis virus; 6: porcine foot-and-mouth disease virus; 7: porcine epidemic diarrhea virus; 8: swine fever virus; 9: porcine circovirus type 2; 10: salmonella suis; 11: porcine pseudorabies virus; 12: negative control.
FIG. 2 is a graph showing the specificity of the porcine reproductive and respiratory syndrome virus L1 line compared to the L3, L5 and L8 lines, wherein 1: l1 is CHSX1401 virus; 2: the L3 strain QYYZ virus; 3: the L5 strain VR2332 virus; 4: l8 is JXA1 strain virus; 5: negative control.
FIG. 3 shows the specific detection of porcine reproductive and respiratory syndrome virus L3 and L1, L5 and L8, wherein 1: the L3 strain QYYZ virus; 2: l1 is CHSX1401 virus; 3: the L5 strain VR2332 virus; 4: l8 is JXA1 strain virus; 5: negative control.
FIG. 4 shows the specific detection of porcine reproductive and respiratory syndrome virus L5 and L1, L3 and L8, wherein 1: the L5 strain VR2332 virus; 2: l1 is CHSX1401 virus; 3: the L3 strain QYYZ virus; 4: l8 is JXA1 strain virus; 5: negative control.
FIG. 5 shows the specific detection of porcine reproductive and respiratory syndrome virus L8 and L1, L3 and L5, wherein 1: l8 is JXA1 strain virus; 2: l1 is CHSX1401 virus; 3: the L3 strain QYYZ virus; 4: the L5 strain VR2332 virus; 5: negative control.
FIG. 6 is a graph showing the results of a sensitivity test for detecting a primer probe used in the L1 system, wherein 1: 1X 10 6 copies/uL；2：1×10 5 copies/uL；3：1×10 4 copies/uL；4：1×10 3 copies/uL；5：1×10 2 copies/uL；6：1×10 1 copies/uL；7：1copies/uL；8：10 -1 cobies/uL; 9: negative control.
FIG. 7 is a standard curve for sensitivity detection of porcine reproductive and respiratory syndrome virus L1 line.
FIG. 8 is a sensitivity test of porcine reproductive and respiratory syndrome virus L3 line. In the figure, 1: 1X 10 6 copies/uL；2：1×10 5 copies/uL；3：1×10 4 copies/uL；4：1×10 3 copies/uL；5：1×10 2 copies/uL；6：1×10 1 copies/uL；7：1copies/uL；8：10 -1 cobies/uL; 9: negative control.
FIG. 9 is a standard curve for sensitivity detection of porcine reproductive and respiratory syndrome virus L3 line.
FIG. 10 is a sensitivity test of porcine reproductive and respiratory syndrome virus L5 line. In the figure, 1: 1X 10 6 copies/uL；2：1×10 5 copies/uL；3：1×10 4 copies/uL；4：1×10 3 copies/uL；5：1×10 2 copies/uL；6：1×10 1 copies/uL；7：1copies/uL；8：10 -1 cobies/uL; 9: negative control.
FIG. 11 is a standard curve for sensitivity detection of porcine reproductive and respiratory syndrome virus L5 line.
FIG. 12 is a sensitivity test of porcine reproductive and respiratory syndrome virus L8 line. In the figure, 1: 1X 10 6 copies/uL；2：1×10 5 copies/uL；3：1×10 4 copies/uL；4：1×10 3 copies/uL；5：1×10 2 copies/uL；6：1×10 1 copies/uL；7：1copies/uL；8：10 -1 cobies/uL; 9: negative control.
FIG. 13 is a standard curve for sensitivity detection of porcine reproductive and respiratory syndrome virus L8 line.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 primer probe design
According to the KP861625.1 gene, JQ308798.1 gene, AY150564.1 gene and EF112445.1 gene sequences of pig breeding and respiratory syndrome registered in Genbank, respectively designing primer probes, wherein the designed primer probe sequences are shown in Table 1 and are synthesized by gene synthesis companies.
Table 1 primer probes for PCR reactions selected
| Primer/probe name | Primer/probe sequence (5 '-3') |
| PRRSV L1-F | TGTCCAGTCATAATCTGGTGTGGYC(SEQ ID NO.1) |
| PRRSV L1-R | GTGGRGGYTTATAYGGRACYTGYYT(SEQ ID NO.2) |
| PRRSV L1-P | FAM-CRGGTTCYRGRTCCT-MGB(SEQ ID NO.3) |
| PRRSV L3-F | CAAAACTTGCACCCCATTG(SEQ ID NO.4) |
| PRRSV L3-R | TGTAACCGGCGCCAATG(SEQ ID NO.5) |
| PRRSV L3-P | VIC-GCCAGACCGACTAGTAGCCA-MGB(SEQ ID NO.6) |
| PRRSV L5-F | GGCTATATGGTGGGCCCTT(SEQ ID NO.7) |
| PRRSV L5-R | CGCCCTTAACAAATTTTGTGAG(SEQ ID NO.8) |
| PRRSV L5-P | CY5-CTAGGCACTCCTGGGGTC-MGB(SEQ ID NO.9) |
| PRRSV L8-F | GGCGAMTCAGACAACCRAMC(SEQ ID NO.10) |
| PRRSV L8-R | ATGAGCCCATATTCTTCYAGR(SEQ ID NO.11) |
| PRRSV L8-P | ROX-TTGGRCAARGACTCG-MGB(SEQ ID NO.12) |
EXAMPLE 2 porcine reproductive and respiratory syndrome Virus L1, L3, L5, L8 quadruple fluorescence PCR
This example provides reagent preparation for the four fluorescent PCR lineages of porcine reproductive and respiratory syndrome virus L1, L3, L5, L8, as shown in Table 2.
Table 2 formulation of reagents
The detection method of the kit provided by the embodiment comprises the following steps:
extraction of genome: extracting viral genome according to the DNA/RNA extraction kit operation instruction, and preserving at-20deg.C for use;
to the PCR reaction tube were added 0.05. Mu.l each of the upstream and downstream primers and probe (100. Mu.M) of PRRSV L1, L3, L5 and L8, 1. Mu.l of enzyme, 10. Mu.l of reaction solution, 3.4. Mu.l of sterile water, 5. Mu.l of template, and a total of 20. Mu.l of RT-PCR amplification system.
Fluorescent PCR reaction procedure: reverse transcription is carried out for 5min at 50 ℃; pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s and annealing at 60℃for 30s were performed for 40 cycles in total.
Example 3 specificity experiments
1. This example provides a specificity test against other swine diseases
The specificity of the primer probe of the present invention was verified by detecting porcine reproductive and respiratory syndrome L1-series CHSX1401 strain virus gene, L3-series QYYZ strain virus gene, L5-series VR2332 strain virus gene, L8-series JXA1 strain virus gene, transmissible gastroenteritis virus (TGEV), porcine foot-and-mouth disease virus (FMDV), porcine Epidemic Diarrhea Virus (PEDV), swine fever virus (CSFV), porcine circovirus type 2 (PCV 2), porcine salmonella (Salm), porcine pseudorabies virus (PRV) and non-nuclease water as negative controls according to the method of example 2.
Genomic DNA/RNA of CHSX1401, QYYZ, VR2332, JXA1, TGEV, FMDV, PEDV, CSFV, PCV2, salm and PRV were extracted using a DNA/RNA extraction kit, and fluorescent PCR amplification assay was performed using the extracted genomic DNA/RNA as a template, and detected as in example 2.
Reaction conditions: fluorescent PCR reaction procedure: reverse transcription is carried out for 5min at 50 ℃; pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s and annealing at 60℃for 30s, followed by 40 cycles in total;
the reaction system: each of the PRRSV L1, L3, L5, L8 upstream and downstream primers and probes (100. Mu.M) was 0.05. Mu.l, 1. Mu.l of the enzyme, 10. Mu.l of the reaction solution, 3.4. Mu.l of sterile water, 5. Mu.l of the template, and 20. Mu.l of the total RT-PCR amplification system.
The results show that: the kit detects CHSX1401, QYYZ, VR2332, JXA1, TGEV, FMDV, PEDV, CSFV, PCV, salm and PRV, and the detection results are not collinear except for the amplification of CHSX1401, QYYZ, VR2332 and JXA1, and the results are shown in figure 1. The detection method is not cross-reacted with other porcine viruses, and the kit has good specificity.
2. Specificity test of L1 series and L3, L5 and L8 series
The specificity of the primer probe of the present invention was verified by detecting the porcine reproductive and respiratory syndrome L1-series CHSX1401 strain virus gene, L3-series QYYZ strain virus gene, L5-series VR2332 strain virus gene, and L8-series JXA1 strain virus gene according to the method of example 2, and using nuclease-free water as a negative control.
Genomic RNA of CHSX1401, QYYZ, VR2332 and JXA1 was extracted using an RNA extraction kit, and fluorescent PCR amplification detection was performed using the extracted genomic RNA as a template, and the detection was performed as in example 2.
Reaction conditions: fluorescent PCR reaction procedure: reverse transcription is carried out for 5min at 50 ℃; pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s and annealing at 60℃for 30s, followed by 40 cycles in total;
the reaction system: each of the PRRSV L1, L3, L5, L8 upstream and downstream primers and probes (100. Mu.M) was 0.05. Mu.l, 1. Mu.l of the enzyme, 10. Mu.l of the reaction solution, 3.4. Mu.l of sterile water, 5. Mu.l of the template, and 20. Mu.l of the total RT-PCR amplification system.
The results show that: the kit detects CHSX1401, QYYZ, VR2332 and JXA1, and the detection results are not on line except for the amplification of the L1-series (FAM channel) CHSX1401 strain, as shown in figure 2. The L1 system is proved to have no cross reaction with the L3, L5 and L8 systems, and the kit has good specificity.
3. Specificity test of L3 series and L1, L5 and L8 series
The specificity of the primer probe of the present invention was verified by detecting the porcine reproductive and respiratory syndrome L1-series CHSX1401 strain virus gene, L3-series QYYZ strain virus gene, L5-series VR2332 strain virus gene, and L8-series JXA1 strain virus gene according to the method of example 2, and using nuclease-free water as a negative control.
Genomic RNA of CHSX1401, QYYZ, VR2332 and JXA1 was extracted using an RNA extraction kit, and fluorescent PCR amplification detection was performed using the extracted genomic RNA as a template, and the detection was performed as in example 2.
Reaction conditions: fluorescent PCR reaction procedure: reverse transcription is carried out for 5min at 50 ℃; pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s and annealing at 60℃for 30s, followed by 40 cycles in total;
the reaction system: each of the PRRSV L1, L3, L5, L8 upstream and downstream primers and probes (100. Mu.M) was 0.05. Mu.l, 1. Mu.l of the enzyme, 10. Mu.l of the reaction solution, 3.4. Mu.l of sterile water, 5. Mu.l of the template, and 20. Mu.l of the total RT-PCR amplification system.
The results show that: the kit provided by the invention detects CHSX1401, QYYZ, VR2332 and JXA1, and the detection results are not on line except for the amplification of L3-series (VIC channel) QYYZ strain, as shown in figure 3. The L3 system is proved to have no cross reaction with the L1, L5 and L8 systems, and the kit has good specificity.
4. Specificity test of L5 series and L1, L3 and L8 series
The specificity of the primer probe of the present invention was verified by detecting the porcine reproductive and respiratory syndrome L1-series CHSX1401 strain virus gene, L3-series QYYZ strain virus gene, L5-series VR2332 strain virus gene, and L8-series JXA1 strain virus gene according to the method of example 2, and using nuclease-free water as a negative control.
Genomic RNA of CHSX1401, QYYZ, VR2332 and JXA1 was extracted using an RNA extraction kit, and fluorescent PCR amplification detection was performed using the extracted genomic RNA as a template, and the detection was performed as in example 2.
Reaction conditions: fluorescent PCR reaction procedure: reverse transcription is carried out for 5min at 50 ℃; pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s and annealing at 60℃for 30s, followed by 40 cycles in total;
the reaction system: each of the PRRSV L1, L3, L5, L8 upstream and downstream primers and probes (100. Mu.M) was 0.05. Mu.l, 1. Mu.l of the enzyme, 10. Mu.l of the reaction solution, 3.4. Mu.l of sterile water, 5. Mu.l of the template, and 20. Mu.l of the total RT-PCR amplification system.
The results show that: the kit provided by the invention detects CHSX1401, QYYZ, VR2332 and JXA1, and the detection results are not on line except for the amplification of the L5 line (CY 5 channel) VR2332 strain, as shown in figure 4. The L5 system is proved to have no cross reaction with the L1, L3 and L8 systems, and the kit has good specificity.
5. Specificity test of L8 series and L1, L3 and L5 series
The specificity of the primer probe of the present invention was verified by detecting the porcine reproductive and respiratory syndrome L1-series CHSX1401 strain virus gene, L3-series QYYZ strain virus gene, L5-series VR2332 strain virus gene, and L8-series JXA1 strain virus gene according to the method of example 2, and using nuclease-free water as a negative control.
Genomic RNA of CHSX1401, QYYZ, VR2332 and JXA1 was extracted using an RNA extraction kit, and fluorescent PCR amplification detection was performed using the extracted genomic RNA as a template, and the detection was performed as in example 2.
Reaction conditions: fluorescent PCR reaction procedure: reverse transcription is carried out for 5min at 50 ℃; pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s and annealing at 60℃for 30s, followed by 40 cycles in total;
the reaction system: each of the PRRSV L1, L3, L5, L8 upstream and downstream primers and probes (100. Mu.M) was 0.05. Mu.l, 1. Mu.l of the enzyme, 10. Mu.l of the reaction solution, 3.4. Mu.l of sterile water, 5. Mu.l of the template, and 20. Mu.l of the total RT-PCR amplification system.
The results show that: the kit provided by the invention detects CHSX1401, QYYZ, VR2332 and JXA1, and the detection results are not on line except for the amplification of L8-series (ROX channel) JXA1 strain, as shown in FIG. 5. The L8 system is proved to have no cross reaction with the L1, L3 and L5 systems, and the kit has good specificity.
Example 4 sensitivity test
The concentration was 1X 10 7 The copies/. Mu.L of the L1, L3, L5, L8 viral plasmids were subjected to 10-fold gradient dilution. Taking 1×10 6 copies/μL～10 -1 COPIES. Mu.L, plasmid of each gradient concentration of L1, L3, L5, L8 virus as template, no nucleaseWater was used as a negative control for the sensitivity detection of the fluorescent RT-PCR method of the invention. The reaction conditions and the reaction system are the same as above.
The sensitivity test result of the primer probe used for the L1 system provided by the invention is shown in figure 6, and the sensitivity test standard curve of the porcine reproductive and respiratory syndrome virus L1 system is shown in figure 7.
The sensitivity test result of the primer probe for the L3 system provided by the invention is shown in figure 8, and the sensitivity test standard curve of the porcine reproductive and respiratory syndrome virus L3 system is shown in figure 9.
The sensitivity test result of the primer probe for the L5 line provided by the invention is shown in figure 10, and the sensitivity test standard curve of the porcine reproductive and respiratory syndrome virus L5 line is shown in figure 11.
The sensitivity test result of the primer probe for the L8 line provided by the invention is shown in figure 12, and the sensitivity test standard curve of the porcine reproductive and respiratory syndrome virus L8 line is shown in figure 13.
The experimental results show that the lowest detection concentration of the L1, L3, L5 and L8 systems is 1 copies/. Mu.L by adopting the kit provided by the invention, the sensitivity is high, and the results are shown in Table 3.
TABLE 3 sensitivity assay for kit
| Sample name | FAM Ct value | VIC Ct value | CY5 Ct value | ROX Ct value |
| 1×10 6 copies/μL | 18.73 | 18.24 | 18.8 | 17.96 |
| 1×10 5 copies/μL | 21.87 | 22.37 | 22.33 | 21.51 |
| 1×10 4 copies/μL | 25.10 | 26.93 | 26.69 | 24.87 |
| 1×10 3 copies/μL | 29.55 | 28.63 | 28.89 | 28.41 |
| 1×10 2 copies/μL | 31.57 | 30.76 | 31.09 | 31.55 |
| 1×10 1 copies/μL | 35.51 | 35.65 | 35.51 | 35.58 |
| 1copies/μL | 38.36 | 38.53 | 38.58 | 38.83 |
| 10 -1 copies/μL | Without any means for | Without any means for | Without any means for | Without any means for |
| Negative control | Without any means for | Without any means for | Without any means for | Without any means for |
Example 5 repeatability test
3 samples of known positives were selected for each of the intra-batch and inter-batch replicates. Repeated detection in batch: 3 known positive samples were run in the same batch of experiments, with 3 replicates per sample. Experiments were repeated between batches: 3 known positive samples were tested in batches, each sample was tested separately, and each sample was set up with 3 replicates. The reaction conditions and the reaction system are the same as above.
The intra-batch variation coefficient of the genes of the porcine reproductive and respiratory syndrome L1, L3, L5 and L8 is between 0.04 and 0.95 percent, and the inter-batch variation coefficient is between 0.17 and 1.04 percent. The results show that the kit of the invention has good repeatability, and the results are shown in Table 4.
Table 4 kit reproducibility assay
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1. The kit for distinguishing the porcine reproductive and respiratory syndrome virus L1, L3, L5 and L8 series strains is characterized by comprising four pairs of primers as follows:
primer pair one:
TGTCCAGTCATAATCTGGTGTGGYC（SEQ ID NO.1），
GTGGRGGYTTATAYGGRACYTGYYT (SEQ ID NO. 2); the probe matched with the primer pair one is: FAM-CRGGTTCYRGRTCCT-MGB;
primer pair two:
CAAAACTTGCACCCCATTG（SEQ ID NO.4），
TGTAACCGGCGCCAATG (SEQ ID NO. 5); the probes matched with the primer pair II are as follows: VIC-GCCAGACCGACTAGTAGCCA-MGB;
primer pair III:
GGCTATATGGTGGGCCCTT（SEQ ID NO.7），
CGCCCTTAACAAATTTTGTGAG (SEQ ID NO. 8); the probes matched with the primer pair III are as follows: CY5-CTAGGCACTCCTGGGGTC-MGB;
primer pair four:
GGCGAMTCAGACAACCRAMC（SEQ ID NO.10），
ATGAGCCCATATTCTTCYAGR (SEQ ID NO. 11); the probes matched with the primer pair IV are as follows: ROX-TTGGRCAARGACTCG-MGB.
2. The kit according to claim 1, wherein the amount of each primer pair and each probe to be matched in the PCR system is the same when the kit is used for quadruple fluorescent PCR amplification.
3. Use of the kit of any one of claims 1-2 for distinguishing between porcine reproductive and respiratory syndrome virus strain L1, CHSX1401, strain L3, QYYZ, strain L5, VR2332 or strain L8, JXA1 for non-disease diagnosis and treatment purposes.
4. A method for distinguishing porcine reproductive and respiratory syndrome virus L1-series CHSX1401 strain, L3-series QYYZ strain, L5-series VR2332 strain or L8-series JXA1 strain for non-disease diagnosis and treatment purposes, characterized in that the primers and probes in the kit according to any one of claims 1-2 are used for performing fluorescent PCR amplification by using porcine reproductive and respiratory syndrome virus as a template, and the porcine reproductive and respiratory syndrome virus strain is judged by using different probes.
5. The method according to claim 4, wherein the ratio of the primer pair and the probe to the template of porcine reproductive and respiratory syndrome virus is (2.5-3): (20-25).
6. The method of claim 5, wherein the PCR reaction is performed as follows: reverse transcription at 48-51 ℃ for 5-8min, pre-denaturation at 92-98 ℃ for 25-30s, denaturation at 92-98 ℃ for 3-5s, annealing at 58-62 ℃ for 25-30s, and carrying out 35-40 cycles.
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| CN102140551A (en) * | 2011-03-25 | 2011-08-03 | 中国动物疫病预防控制中心 | General real-time fluorescent RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) method and kit for detecting porcine reproductive and respiratory syndrome virus |
| CN105648119A (en) * | 2016-03-30 | 2016-06-08 | 广东温氏食品集团股份有限公司 | Primer, kit and method for detecting porcine reproductive and respiratory syndrome viruses |
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| CN106435039A (en) * | 2016-12-26 | 2017-02-22 | 广东温氏食品集团股份有限公司 | Primers, probe, kit and method for detecting PRRS (porcine reproductive and respiratory syndrome) NADC-30 strain |
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| CN102140551A (en) * | 2011-03-25 | 2011-08-03 | 中国动物疫病预防控制中心 | General real-time fluorescent RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) method and kit for detecting porcine reproductive and respiratory syndrome virus |
| CN105648119A (en) * | 2016-03-30 | 2016-06-08 | 广东温氏食品集团股份有限公司 | Primer, kit and method for detecting porcine reproductive and respiratory syndrome viruses |
| CN106119421A (en) * | 2016-08-30 | 2016-11-16 | 广东温氏食品集团股份有限公司 | Fluorescent quantitation detection primer, probe and the test kit of pig blue-ear disease QYYZ strain |
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