CN117327839A - PCR-HRM detection method for porcine epidemic diarrhea virus vaccine strain and wild strain - Google Patents

PCR-HRM detection method for porcine epidemic diarrhea virus vaccine strain and wild strain Download PDF

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CN117327839A
CN117327839A CN202311145046.8A CN202311145046A CN117327839A CN 117327839 A CN117327839 A CN 117327839A CN 202311145046 A CN202311145046 A CN 202311145046A CN 117327839 A CN117327839 A CN 117327839A
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pcr
epidemic diarrhea
porcine epidemic
diarrhea virus
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贺笋
李媛
张盼
潘晓梅
曹剑
杜久斌
张超
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Tiankang Pharmaceutical Co ltd
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Abstract

The invention discloses a PCR-HRM detection method for porcine epidemic diarrhea virus vaccine strain and wild strain, belonging to the technical field of virus detection. The invention designs a pair of novel detection primers for identifying porcine epidemic diarrhea virus vaccine strain and wild strain, wherein the upstream primers are as follows: TTGCGTAAGATGGCACAAC, the downstream primer is: ATAACAGTAT CACCAAGCCATAG. The primer is matched with a high-resolution melting curve technology, so that a high-throughput detection method with high specificity, good sensitivity and high accuracy is developed, and the primer can be used for identifying different strains of porcine epidemic diarrhea virus.

Description

PCR-HRM detection method for porcine epidemic diarrhea virus vaccine strain and wild strain
Technical Field
The invention relates to a PCR-HRM detection method of porcine epidemic diarrhea virus vaccine strain and wild strain, belonging to the technical field of virus detection.
Background
Porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV) belongs to the genus coronavirus of the family coronaviridae, and three proteins exist on the surface of the virion: membrane protein (M), fiber protein (S), envelope protein (E), and only nucleocapsid protein (N) within the virus, these several structural proteins are encoded by the ORF1 gene reading frame. PEDV is a virus commonly prevalent in swine herds and one of the viruses with higher detection positive rate. Porcine epidemic diarrhea (Porcine Epidemic Diarrhea, PED) is an acute, high-contact intestinal infectious disease which is caused by PEDV and takes diarrhea, vomiting and dehydration of piglets as main symptoms, pigs of various ages are susceptible, especially nursing piglets, the morbidity is up to 100%, and the mortality is 60-100%. Up to now, almost all provinces in China report the occurrence and popularity of PED, which seriously affects the healthy development of pig industry in China. The prevention and control of PEDs are mostly performed by vaccines, but there are also animals that fail to be immunized and are infected with PEDV wild strains at the same time as immunization is performed. The identification of the PEDV vaccine strain and the wild strain not only can evaluate whether the farm is infected by the wild strain, but also has important guiding significance for epidemiological investigation of the pig farm and formulation of vaccine immunization programs, and the cost is lower.
There are various methods for detecting PEDV, such as a colloidal gold method for detecting antigen and an ELISA method for detecting antibody, which are easy to operate, but cannot distinguish vaccine strains from wild strains, and an RT-PCR method for detecting nucleic acid, which is mainly based on the S gene or the auxiliary gene ORF3 for detecting PEDV, and the RT-PCR method is cumbersome to operate, takes a long time, can be uncapped back and forth in the detection process, is easy to cause pollution, and has high cost.
The high-resolution melting curve analysis technique (high-resolution melting, HRM) is a method for Single Nucleotide Polymorphism (SNP) and genotyping research, and is a simple, rapid and low-cost post-PCR amplification detection technique which can be used for high-throughput mutation scanning and genotyping. Although some HRM detection methods for identifying porcine epidemic diarrhea virus vaccine strain and wild strain are reported at present, detection sensitivity, specificity and accuracy still need to be further improved due to the problems of selection of different sites, design of primers and the like.
In view of this, the present invention has been made.
Disclosure of Invention
In order to solve the problems, the invention provides a novel porcine epidemic diarrhea virus vaccine strain and wild strain differential diagnosis detection method based on a high-resolution dissolution curve.
The first object of the invention is to provide a detection primer for porcine epidemic diarrhea virus, the nucleotide sequence of which is shown in SEQ ID NO. 5-6.
The second object of the invention is to provide a method for detecting porcine epidemic diarrhea virus vaccine strain and wild strain, which adopts a PCR-HRM reaction system comprising the detection primer to amplify and detect a sample containing porcine epidemic diarrhea virus.
Further, the amplification reaction procedure was: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 10s, annealing at 52-65 ℃ for 10s, extension at 72 ℃ for 10s, and circulation for 40 times; 95℃60s,40℃60s,65℃1s,97℃1s.
Further, the annealing temperature is preferably 60 ℃.
Further, the PCR-HRM reaction system is as follows: green-2-Go2XqPCR-S Mastermix volume of 8-12. Mu.L, upstream and downstream primers of 0.2-0.4. Mu.L each, temp 1-3. Mu.L, ddH 2 O makes up 18-22. Mu.L.
Preferably, the PCR-HRM reaction system is: green-2-Go2XqPCR-S Mastermix volume of 10. Mu.L, upstream and downstream primers of 0.4. Mu.L each, temp 2. Mu.L, ddH 2 O makes up 20. Mu.L of the reaction system.
Further, the melting curve shows melting peaks at 81.7-81.9 ℃, and the PEDV wild strain is judged; the melting curve shows melting peaks at 82.3-82.5 ℃, and the PEDV vaccine strain is determined.
Further, melting curve analysis was performed at a melting rate of 0.07 ℃/s from 65 ℃ to 97 ℃.
The third object of the invention is to provide a kit for differential diagnosis and detection of porcine epidemic diarrhea virus vaccine strain and wild strain, which comprises: PCR-HRM reaction system containing the above detection primer.
Further, the kit also comprises a positive control substance or/and a negative control substance.
The kit can be used for detecting and identifying porcine epidemic diarrhea virus vaccine strains and wild strains.
The invention has the beneficial effects that:
the primer design is carried out aiming at the base difference of a plurality of sites of wild strains and vaccine strains, and a pair of primers capable of specifically amplifying target genes of the porcine epidemic diarrhea virus vaccine strains and the wild strains is obtained through screening, and the primers are different from target sequences aimed at in the existing detection method, so that the detection sensitivity and the detection accuracy are obviously improved. The PCR-HRM detection method based on the primer pair design has the advantages of low cost, simple operation, high detection speed, high flux, high accuracy, good specificity and good repeatability.
Drawings
FIG. 1 shows the detection limit results of vaccine strains and wild strains according to the invention.
FIG. 2 shows the result of the specific assay in example 5 of the present invention.
FIG. 3 shows qPCR detection results (Taqman method) of 30 clinical samples.
FIG. 4 shows the qPCR assay results (Taqman method) for 20 clinical samples.
FIG. 5 shows the HRM assay results of 30 clinical samples (invention).
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The main reagents and material sources used in the following examples are shown in table 1:
table 1 reagent and manufacturer thereof
Example 1 primer screening
In this example, different primers were designed to compare and verify the specificity of the primers of the present invention. The comparison of the PEDV ORF1 sequences, the wild strain, compared with the vaccine strain, has the following characteristics, at 4405nt site g→a,4440nt, 4446nt, 9021nt, 9048nt site c→t,8994nt site c→a,9054nt site t→c. Based on the mutation characteristics, two pairs of primers were designed for screening (see table 2), and the reaction system: green-2-Go2XqPCR-S Mastermix volume of 10. Mu.L, upstream and downstream primers of 0.2. Mu.L each, temp 2. Mu.L, ddH 2 O makes up 20. Mu.L of the reaction system. The reaction procedure: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 10s, and circulation for 40 times; 95℃60s,40℃60s,65℃1s,97℃1s.
TABLE 2 primer information
Proved by verification, the ORF1-4400-F/R amplification fails, and the ORF1-9000-F1/R1 is successfully amplified and can be used for the establishment of a subsequent method. Wherein the nucleotide sequence of the recombinant plasmid CV777-ORF1-4400 is shown in SEQ ID NO.1, the nucleotide sequence of the recombinant plasmid CH-ORF1-4400 is shown in SEQ ID NO.2, the nucleotide sequence of the recombinant plasmid CV777-ORF1-9000 is shown in SEQ ID NO.3, and the nucleotide sequence of the recombinant plasmid CH-ORF1-9000 is shown in SEQ ID NO.4.
EXAMPLE 2 optimum Tm value screening
The annealing temperature is selected from 52-64.5 deg.c and the annealing temperature with relatively high delta Tm value. Amplifying target genes by adopting primers ORF1-9000-F1/R1, and adopting a reaction system: green-2-Go2XqPCR-SMastermix volumes were 10. Mu.L, and the upstream and downstream primers were 0.2. Mu.L each, temp 2. Mu.L, ddH 2 O makes up 20. Mu.L of the reaction system. Reaction processThe sequence is as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 10s, annealing at 52-64.5 ℃ for 10s, extension at 72 ℃ for 10s, and circulation for 40 times; 95℃60s,40℃60s,65℃1s,97℃1s. The results are shown in Table 3.
TABLE 3Tm results
Since the DeltaTm value is larger than 0 and is preferably larger, it can be seen from Table 3 that the DeltaTm value is at most 0.54, and the corresponding Tm value is 60℃and thus the annealing temperature of 52 to 64.5℃can be used for strain discrimination, and the optimum Tm value is 60 ℃.
Example 3 optimal addition amount screening of primers
The optimal addition amount of the primer is selected, the selection range is 0.2-0.4 uL, and the combination with higher delta Tm value is selected. The reaction system is as follows: 10. Mu.L Green-2-Go2XqPCR-S Mastermix, 2.0. Mu.L Temp, 0.2-0.4. Mu.L ORF1-9000-F1/R1, plus ddH 2 O was made up to 20. Mu.L. The reaction procedure is: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 10s, and circulation for 40 times; 95℃60s,40℃60s,65℃1s,97℃1s. The respective contents of the upstream primer and the downstream primer and the corresponding Δtm values are specifically shown in table 4.
TABLE 4 primer set results
Since the DeltaTm value is larger than 0 and is preferably larger, it can be seen from Table 4 that the highest DeltaTm value is 0.51 and the corresponding primer addition amount is 0.4. Mu.L, and therefore, primer addition amounts of 0.2 to 0.4. Mu.L/each can be used for strain discrimination, wherein the optimal primer addition amounts are each 0.4. Mu.L.
Example 4 sensitivity verification
1. Method of
(1) Extracting porcine epidemic diarrhea virus RNA in a sample by using a Tianlong nucleic acid extraction kit, wherein the sample can be intestinal tissue, anal swab, excrement and cell culture.
(2) Preparation of positive standard: constructing PEDV wild strain and vaccine strain positive standard, providing HRM positive control for detection of subsequent clinical samples, and diluting plasmid DNA to 10 7 The copies/. Mu.L served as positive control.
(3) The operation process comprises the following steps: the preparation reaction system is as follows: 10. Mu.L Green-2-Go2XqPCR-SMasermix, 2.0. Mu.L Temp, 0.4. Mu.L ORF1-9000-F1/R1, add ddH 2 O was made up to 20. Mu.L. The amplification procedure was set as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 10s, and circulation for 40 times; detection was performed at 95℃for 60s,40℃for 60s,65℃for 1s, and 97℃for 1s.
(4) Analysis of results: the analysis process is carried out in a Rogowski fluorescence quantitative PCR instrument, and the instrument can complete the whole process of PCR-HRM analysis.
2. Results
(1) The result judgment criteria were: the melting curve shows melting peaks at 81.7-81.9 ℃, and the PEDV wild strain is judged; the melting curve shows melting peaks at 82.3-82.5 ℃, and the PEDV vaccine strain is determined.
(2) The minimum detection limit of the wild strain/vaccine strain is 5 copies/. Mu.L, see FIG. 1 (the purple area results are detectable, the red area results are undetectable).
(3) The CV values in the groups and between the groups are less than 1%, which shows that the method has good stability. See table below.
TABLE 5 results of repeatability verification
EXAMPLE 5 specific assay
The present example uses a variety of common viruses to perform specificity verification of the methods of the present invention. The viruses used in this example include: TGEV, poRV, PDCoV E.coli, APP, SS, CP, HPS, mhp, CSFV, PRRSV, PCV2, a wild strain of PEDV and a vaccine strain of PEDV. The results are shown in FIG. 2, and no melting curve appears for other pathogens except the PEDV wild strain and the PEDV vaccine strain, which shows that the method has good specificity.
Example 6 comparison of clinical sample detection Effect
30 samples of disease materials collected from Xinjiang scale pig farm are identified and detected, and the comparison is made between the PEDV identification fluorescent quantitative PCR detection method (Taqman probe method) and the method of the invention.
(1) PEDV species identification fluorescent quantitative PCR assay (Taqman)
The primers are shown in Table 6, the reaction system is shown in Table 7, and the amplification procedure is shown in Table 8.
TABLE 6 primer probe sequences (seed identification)
TABLE 7Taqman reaction System
TABLE 8Taqman amplification procedure
The results are shown in FIGS. 3-4. Of the 30 samples, 20 samples with positive detection results are further identified and detected for distinguishing PEDV wild strain from vaccine strain infection. The primer combinations of fluorescent quantitative PCR (Taqman) are shown in Table 9, the reaction system is shown in Table 7, and the reaction procedure is shown in Table 8.
TABLE 9 fluorescence quantitative PCR method primer information
Results 20 samples with positive detection results were wild strains.
(2) The method of the invention detects
The PCR-HRM method is adopted to directly carry out seed identification and parting detection on 30 clinical samples, the used primers are ORF1-9000-F1/R1, and the reaction system is as follows: green-The volume of 2-Go2XqPCR-S Mastermix was 10. Mu.L, each of the upstream and downstream primers was 0.4. Mu.L, temp 2. Mu.L, ddH 2 O makes up 20. Mu.L of the reaction system.
The reaction procedure is: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 10s, and circulation for 40 times; 95℃60s,40℃60s,65℃1s,97℃1s.
The results show (FIG. 5) that 20 samples were obtained as wild strains. Therefore, the method is consistent with the fluorescent quantitative PCR (Taqman) and the identification detection result, and the method can realize identification parting by one-time detection, and has shorter time and lower cost.
(3) Cost accounting contrast
Taking epidemiological investigation and detection sample numbers in Xinjiang as reference examples, 1000 cases are detected, the unit price of one sample detected and typed by the Taqman method is 20 yuan, the unit price of one sample by the HRM method is 3 yuan, the cost of detection and typing by the Taqman method is 4 ten thousand yuan, the cost of detection by the HRM method is 3 kiloyuan, and the cost is reduced by more than 10 times.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims (10)

1. A detection primer for porcine epidemic diarrhea virus is characterized in that the nucleotide sequence is shown as SEQ ID NO. 5-6.
2. A method for detecting porcine epidemic diarrhea virus vaccine strain and wild strain, characterized in that a sample containing porcine epidemic diarrhea virus is amplified and detected by using a PCR-HRM reaction system comprising the detection primer of claim 1.
3. The method according to claim 2, wherein the reaction procedure for amplification using the PCR-HRM reaction system is: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 10s, annealing at 52-65 ℃ for 10s, extension at 72 ℃ for 10s, and circulation for 40 times; 95℃60s,40℃60s,65℃1s,97℃1s.
4. A method according to claim 3, wherein the annealing temperature in the reaction sequence is 60 ℃.
5. The method of claim 2, wherein the PCR-HRM reaction system is: green-2-Go2XqPCR-S Mastermix volume of 8-12. Mu.L, upstream and downstream primers of 0.2-0.4. Mu.L each, temp 1-3. Mu.L, ddH 2 O is added to the mixture to be 18-22 mu L; preferably, the PCR-HRM reaction system is: green-2-Go2XqPCR-S Mastermix volume of 10. Mu.L, upstream and downstream primers of 0.4. Mu.L each, temp 2. Mu.L, ddH 2 O makes up 20. Mu.L of the reaction system.
6. The method of claim 5, wherein each of the upstream and downstream primers is 0.4. Mu.L.
7. The method according to claim 2, characterized in that: the melting curve shows melting peaks at 81.7-81.9 ℃, and the PEDV wild strain is judged; the melting curve shows melting peaks at 82.3-82.5 ℃, and the PEDV vaccine strain is determined.
8. The kit for identifying, diagnosing and detecting the porcine epidemic diarrhea virus vaccine strain and wild strain is characterized by comprising the following components: a PCR-HRM reaction system comprising the detection primer of claim 1.
9. The kit of claim 8, wherein: the kit also comprises a positive reference substance or/and a negative reference substance.
10. Use of the kit according to claim 8 or 9 for detecting porcine epidemic diarrhea virus vaccine strain and wild strain.
CN202311145046.8A 2023-09-06 2023-09-06 PCR-HRM detection method for porcine epidemic diarrhea virus vaccine strain and wild strain Pending CN117327839A (en)

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