阅读说明：本技术 鉴别猪流行性腹泻病毒疫苗株与流行毒株的荧光定量pcr引物、探针、试剂盒及应用 (Fluorescent quantitative PCR primer, probe and kit for identifying porcine epidemic diarrhea virus vaccine strain and epidemic strain and application ) 是由 朱俊辉 廉维 陈立思 何玉友 杨泽彬 王国辉 李长艳 赵涵 贾小营 白杨 郑洪娟 于 2020-01-07 设计创作，主要内容包括：本发明公开了鉴别猪流行性腹泻病毒疫苗株与流行毒株的荧光定量PCR引物对和探针、试剂盒及其应用。本发明筛选获得特异性好的2条探针和1对特异性引物,使用一步法扩增试剂进行扩增,建立了一种可快速鉴别诊断PEDV疫苗株与流行毒株的荧光定量RT-PCR检测试剂盒,只扩增猪流行性腹泻病毒疫苗株和流行株,而对猪传染性胃肠炎病毒、轮状病毒、猪瘟病毒、伪狂犬病毒和猪圆环病毒均无非特异性扩增,保证检测结果的准确性。本发明所建立的检测试剂盒具有较高灵敏度和特异性,同时具有较好的批内和批间检测的稳定性和准确性,可以实现PEDV毒株类型精准鉴定,符合检测标准,能广泛应用于PEDV临床监测和疾病诊断。(The invention discloses a fluorescent quantitative PCR primer pair, a probe and a kit for identifying a porcine epidemic diarrhea virus vaccine strain and an epidemic strain and application thereof. The invention obtains 2 probes with good specificity and 1 pair of specific primers by screening, and uses a one-step amplification reagent for amplification to establish a fluorescent quantitative RT-PCR detection kit capable of quickly identifying and diagnosing PEDV vaccine strains and epidemic strains, only amplifies the porcine epidemic diarrhea virus vaccine strains and the epidemic strains, but has no non-specific amplification to the porcine transmissible gastroenteritis virus, rotavirus, classical swine fever virus, pseudorabies virus and porcine circovirus, thereby ensuring the accuracy of the detection result. The detection kit established by the invention has higher sensitivity and specificity, has better stability and accuracy of detection in batches and among batches, can realize accurate identification of the PEDV strain type, meets the detection standard, and can be widely applied to PEDV clinical monitoring and disease diagnosis.)

1. The fluorescent quantitative PCR primer pair and the probe for identifying the porcine epidemic diarrhea virus vaccine strain and the epidemic strain are characterized in that the primer pair consists of two primers shown by SEQ ID No.1 and SEQ ID No.2, and the probe is a single probe with a nucleotide sequence shown by SEQ ID No.3 or SEQ ID No.4 or a probe pair consisting of two probes with nucleotide sequences shown by SEQ ID No.3 and SEQ ID No.4 respectively.

2. The pair of fluorescent quantitative PCR primers and the probe according to claim 1, wherein the two ends of the probe having the nucleotide sequence represented by SEQ ID No.3 are connected with FAM fluorescent group and TAMAR quenching group, respectively; two ends of the probe with the nucleotide sequence shown in SEQ ID No.4 are respectively connected with a HEX fluorescent group and a TAMAR quenching group.

3. The pair of fluorescent quantitative PCR primers and probe as claimed in claim 2, wherein the probe having the nucleotide sequence represented by SEQ ID No.3 has a FAM fluorescent group attached to its 5 'end and a TAMAR quenching group attached to its 3' end.

4. The pair of fluorescent quantitative PCR primers and probe as claimed in claim 2, wherein the probe having the nucleotide sequence represented by SEQ ID No.4 has HEX fluorescent group connected to its 5 'end and TAMAR quenching group connected to its 3' end.

5. A fluorescent quantitative PCR kit for identifying porcine epidemic diarrhea virus vaccine strains and epidemic strains comprises an RNA reverse transcription reagent, a fluorescent quantitative RT-PCR amplification reagent, a fluorescent quantitative PCR primer pair and a probe; the method is characterized in that the fluorescent quantitative PCR primer pair and the probe are the fluorescent quantitative PCR primer pair and the probe in any one of claims 1 to 4.

6. Use of the fluorescent quantitative PCR primer pair and the probe as set forth in any one of claims 1 to 4 for the preparation of a reagent or a medicament for diagnosing or differentiating the porcine epidemic diarrhea virus vaccine strain and the epidemic strain.

Technical Field

The invention relates to identification of a porcine epidemic diarrhea virus vaccine strain and an epidemic strain, and further relates to a PCR primer pair and a probe for identifying the porcine epidemic diarrhea virus vaccine strain and the epidemic strain and a fluorescent quantitative RT-PCR detection kit, belonging to the field of molecular identification of the porcine epidemic diarrhea virus vaccine strain and the epidemic strain.

Background

PEDV is a highly contact infectious disease with acute diarrhea of piglets as a clinical characteristic caused by Porcine Epidemic Diarrhea Virus (PEDV). the PEDV is a member of the family Coronaviridae, α genus coronaviruses, a enveloped single-stranded positive-strand RNA virus with a genome of 28kb in full length and seven open reading frames, encodes 4 structural proteins (S, E, M and N) and 3 non-structural proteins (ORF1a, ORF1b and ORF3), wherein the spike protein encoded by the S gene is an important component of PEDV, which is closely related to the production of neutralizing antibodies, viral infection and receptor fusion, and is an important protein for the research and development of current pathogenic mechanisms, diagnostic reagents and vaccines, and the PEDV is an important protein for the development of the world pig industry from 1971, causing huge economic loss, the year-scale of PEDV in China, the year-year.

Currently, PEDV detection methods include RT-PCR, E L ISA, IFA, RT-L AMP and other detection methods, but the methods are difficult to realize accurate and rapid detection and diagnosis, and meanwhile, the methods are difficult to accurately identify vaccine strains and epidemic strains in the current pig farm.

Disclosure of Invention

The invention aims to provide a fluorescent quantitative PCR primer pair and a probe for identifying a porcine epidemic diarrhea virus vaccine strain and an epidemic strain;

the invention also aims to provide a fluorescent quantitative RT-PCR detection kit for identifying the porcine epidemic diarrhea virus vaccine strain and the epidemic strain.

The above object of the present invention is achieved by the following technical solutions:

according to the difference of multiple bases of the porcine epidemic diarrhea virus vaccine strain and the epidemic strain at the N end of the S gene, multiple pairs of probes for detecting the vaccine strain and the epidemic strain are respectively designed, through repeated test verification and screening, two probes capable of simultaneously, stably and effectively detecting the porcine epidemic diarrhea virus vaccine strain and the epidemic strain are finally obtained, a pair of upstream and downstream primers is designed between 110bp and 170bp, and a one-step amplification reagent is used for amplification, so that the fluorescent quantitative RT-PCR detection kit capable of quickly identifying and diagnosing the PEDV vaccine strain and the epidemic strain is established, and the kit can be used for quickly detecting the porcine epidemic diarrhea virus in a breeding farm and distinguishing and judging the immune effect of the vaccine.

The invention firstly provides a pair of fluorescent quantitative RT-PCR primers for identifying porcine epidemic diarrhea virus vaccine strains and epidemic strains, wherein the primer pair respectively consists of two base sequences shown in SEQ ID No.1 and SEQ ID No.2, and the amplified PEDV fragment has a length of 135 bp:

SEQ ID No.1：5’-AYTTTAGGCGGTTCTTTTC-3’；

SEQ ID No.2：5’-ARATACCATGAACGCCACT-3’；

the invention further provides 8 porcine epidemic diarrhea virus vaccine strains and an epidemic strain identification fluorescent quantitative RT-PCR probe, wherein the probe is a single probe with a nucleotide sequence shown in SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 and SEQ ID No. 10; or any pair of probes selected from (1) - (4): (1) SEQ ID No.3 and SEQ ID No. 4; (2) SEQ ID No.5 and SEQ ID No. 6; (3) SEQ ID No.7 and SEQ ID No. 8; (4) SEQ ID No.9 and SEQ ID No. 10.

SEQ ID No.3：FAM-GCCGTCGTYGYTTTGGGTGGTT-TAMRA；

SEQ ID No.4：HEX-GCAGTTGTYGYACTGGGCGGTT-TAMRA。

SEQ ID No.5：CY5-TAGCTGGTACTGTGGCACAGGCAT-TAMRA

SEQ ID No.6：HEX-TCAACTTGGTACTGTGCTGGCCA-TAMRA

SEQ ID No.7：FAM-TAGCCATATTAGGAGGTGGTCAGG-BHQ

SEQ ID No.8：HEX-TTSTCAGCTACATCGATTCT-TAMRA

SEQ ID No.9：CY5-CCTAGTATGAACTCTTCTAGCT-TAMRA

SEQ ID No.10：FAM-ATTGGTGAAAACAGGGTGTC-TAMRA

The invention discovers that: a single probe shown in SEQ ID No.3 or SEQ ID No.4 or a fluorescent quantitative RT-PCR probe consisting of two probes with nucleotide sequences shown in SEQ ID No.3 and SEQ ID No.4 respectively, which can stably and effectively detect the porcine epidemic diarrhea virus vaccine strain and the epidemic strain;

the two ends of the probe with the nucleotide sequence shown in SEQ ID No.3 are respectively connected with a FAM fluorescent group and a TAMAR quenching group; two ends of the probe with the nucleotide sequence shown as SEQ ID No.4 are respectively connected with a HEX fluorescent group and a TAMAR quenching group. Wherein, the 5 'end of the probe with the nucleotide sequence shown as SEQ ID No.3 is connected with a FAM fluorescent group, and the 3' end is connected with a TAMAR quenching group; the 5 'end of the probe with the nucleotide sequence shown in SEQ ID No.4 is connected with a HEX fluorescent group, and the 3' end of the probe is connected with a TAMAR quenching group.

The invention further provides a kit for identifying the porcine epidemic diarrhea virus vaccine and the epidemic strain by the fluorescent quantitative RT-PCR, which comprises an RNA reverse transcription reagent, a fluorescent quantitative RT-PCR amplification reagent, a fluorescent quantitative PCR primer pair and a probe.

The RT-qPCR MIX amplification reagent comprises One-Step PrimeScript III RT-qPCR MIX (2 ×) and ROX Reference Dye II (50 ×).

The fluorescent quantitative PCR primer pair consists of SEQ ID No.1 and SEQ ID No. 2; the fluorescent quantitative PCR probe is a probe pair consisting of a single probe shown by SEQ ID No.3 or SEQ ID No.4 or two probes with nucleotide sequences shown by SEQ ID No.3 and SEQ ID No.4 respectively.

The invention further provides a method for applying the kit for identifying the fluorescent quantitative RT-PCR detection of the porcine epidemic diarrhea virus vaccine and the epidemic strain, which comprises the following steps:

(1) extracting RNA of a sample to be detected;

(2) performing fluorescent quantitative RT-PCR reaction on RNA extracted from a sample by using a one-step amplification method;

(3) judging according to the fluorescent quantitative RT-PCR reaction result, wherein the specific judgment process is that when an S-shaped amplification curve appears on the FAM detection probe and the Ct value is less than 35 and neither the HEX probe nor the negative control is amplified, the detection sample is the PEDV epidemic strain; when the HEX detection probe has an S-type amplification curve and the Ct value is less than 35, and neither the FAM probe nor the negative control is amplified, the detection sample is a PEDV vaccine strain; when the FAM probe and the HEX are simultaneously amplified to form an S-shaped curve, the negative control is not amplified, and a detection sample is that the PEDV vaccine strain and the epidemic strain are simultaneously infected; and when the CT value of the amplification curves of the FAM probe and the HEX probe is more than 35 and less than or equal to 40, judging that the result is suspicious, re-detecting, and judging that the result is negative if the re-detection result is still suspicious.

The fluorescent quantitative RT-PCR amplification system is 15 mu L One-Step PrimeScript III RT-qPCRMix (2 ×), ROX Reference Dye or Dye II (50 ×)1 mu L, primers SEQ ID No.1 and SEQ ID No.2 respectively account for 1 mu L, probes SEQ ID No.3 and SEQ ID No.4 respectively account for 1 mu L, and ddH2O 3 mu L template 2 mu L, and the total amount is 25 mu L.

The fluorescent quantitative RT-PCR amplification reaction program comprises the following steps: reverse transcription is carried out at 52 ℃ for 5min and at 95 ℃ for 10s, the PCR process is carried out at 95 ℃ for 5s and at 60 ℃ for 30s, and the total number of PCR reaction cycles is 40.

The main beneficial effects of the invention include:

1. the invention designs 4 pairs of probes and 1 pair of primers by screening PEDV vaccine strains and epidemic strains published in NCBI GenBank at multiple variation positions of an S gene sequence, and establishes a fluorescent quantitative RT-PCR detection method of the porcine epidemic diarrhea virus vaccine strains and the epidemic strains.

2. The 2 probes and 1 pair of detection primers obtained by final screening have good specificity, only porcine epidemic diarrhea virus vaccine strains and epidemic strains are amplified, and non-specific amplification is not performed on porcine transmissible gastroenteritis virus, rotavirus, classical swine fever virus, pseudorabies virus and porcine circovirus, so that the accuracy of a detection result is ensured.

3. The invention develops and establishes a detection kit for the PEDV vaccine strain and the epidemic strain, realizes the accurate identification of the type of the PEDV strain, meets the detection standard, and can be widely applied to PEDV clinical monitoring and disease diagnosis.

Drawings

FIG. 1 is a graph of the results of various fluorescent probe amplification screens, 1: SEQ ID No. 3; 2: SEQ ID No. 4; 3: SEQ ID No. 7; 5: SEQ ID No. 5; 6: SEQ ID No. 6; in the figure 7: SEQ ID No. 8; in the figure 8: SEQ ID No. 9; in the figure 9: SEQ ID No. 10.

FIG. 2 shows the identification result of fluorescent quantitative RT-PCR PEDV epidemic strain, 1: an epidemic strain of porcine epidemic diarrhea virus; 2: porcine epidemic diarrhea virus vaccine strains.

Fig. 3 is the results of fluorescent quantitative RT-PCR PEDV vaccine strain identification, 1: an epidemic strain of porcine epidemic diarrhea virus; 2: porcine epidemic diarrhea virus vaccine strains.

FIG. 4 is the result of fluorescent quantitative RT-PCR to identify both PEDV vaccine strains and circulating strains simultaneously, 1: an epidemic strain of porcine epidemic diarrhea virus; 2: porcine epidemic diarrhea virus vaccine strains.

FIG. 5 shows the result of the fluorescent quantitative RT-PCR specificity assay, 1: an epidemic strain of porcine epidemic diarrhea virus; 2: porcine epidemic diarrhea virus vaccine strains; 3: transmissible gastroenteritis virus of swine; 4: hog cholera virus; 5: rotavirus; 6; porcine pseudorabies virus; 7: porcine circovirus type 2; 8, negative control.

FIG. 6 shows the result of fluorescent quantitative RT-PCR sensitivity assay, in which 1: the concentration of porcine epidemic diarrhea virus nucleic acid is 1.53 × 10⁶copies/μL；2：1.53×10⁵copies/μL；3：1.53×10⁴copies/μL； 4：1.53×10³copies/μL；5：1.53×10²copies/μL；6：1.53×10¹copies/μ L, 7: 1.53copies/μ L, 8: negative control.

FIG. 7 fluorescent quantitative RT-PCR repeatability test identification results, 1: 10⁷copies/μL；2：10⁶copies/μL；3：10⁵copies/μL；4：10⁴copies/μL

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.