RT-PCR kit for identification and diagnosis of porcine epidemic diarrhea virus and application of RT-PCR kit

[0033] Example 1 Detection of piglet diarrhea samples and analysis of ORF3 gene sequence of epidemic strain

[0034] (1) Primer design

[0035] According to PEDV ZJCZ-4 (GenBank sequence number: JX524137), a pair of primers ORF3-P1/ORF3-P2 were designed and synthesized to amplify the full ORF3 gene. With reference to the ORF3 gene sequence of PEDV CV777 (GenBank sequence number: AF353511), ZJCZ-4 and vaccine strain DR13 (GenBank sequence number: EU054930), a pair of identification primers ORF3-JD1/ORF3-JD2 was designed and synthesized, and the identification primers were located at ORF3 The conserved regions at both ends of the deletion region (245nt~294nt), whose amplification range covers the entire deletion region. The above two pairs of primers are shown in Table 1. All primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

[0036] Table 1 Amplification of PEDV ORF3 gene primers and identification primers

[0037]

[0038] (2) RNA extraction and reverse transcription from samples

[0039] Centrifuge the piglet's diarrhea samples collected and stored in our laboratory. Take the PEDV isolates HLJ-1 strain and ZJCZ4 strain and 300μL each of the processed clinical samples, and extract viral RNA according to the RNeasy Plus Mini Kit instructions. At the same time, according to the RevertAidTM First Strand cDNA Synthesis Kit instructions, the RNA extracted from the sample was reverse transcribed into single-stranded cDNA, that is, the total RNA of the extracted sample was 11μL, random primer Oligo (dT) 1μL, 5× buffer 4μL, RNase inhibitor (20u /μL) 1μL, dNTP Mix (10mM) 2μL, M-MuLV reverse transcriptase (200u/μL) 1μL, after cDNA synthesis, store at -20℃ for later use.

[0040] (3) Amplification, cloning and sequencing of ORF3 gene in clinical samples

[0041] Eleven PEDV positive samples (ZZ-1, HEB-1, FQ1-5, QZ1-4, SD-2, SD-4, SD-5, SDXY-7B, etc.) were selected from the piglet diarrhea samples kept in our laboratory. BJ-26, ZJ-2, ZJ-3), using ORF3-P1/P2 primers to amplify and clone the ORF3 gene, and sequenced by Invitrogen. Using MEGA4.1 software, the ORF3 gene of the above sample strains were compared and analyzed with the PEDV ORF3 gene reference sequence published on GenBank (see Table 2 for the reference strain).

[0042] Table 2 Names and sources of 12 PEDV reference strains used for ORF3 gene sequence analysis

[0043] Isolate name

[0044] (4) Results: Sequence analysis of ORF3 gene of epidemic strain

[0045] The present invention cloned and obtained the ORF3 genes of 11 PEDV positive samples. After sequencing, the ORF3 gene sequences of 11 samples were analyzed by MEGA 4.1. The results showed that 11 samples were compared with the reference sequences provided by GenBank listed in Table 2. Nine of the epidemic strains had no gene deletion in the ORF3 gene, and the other two strains (HEB-1 and SD-5) had 49 base deletions between 245nt and 293nt (such as figure 1 As shown), the nucleotide homology of 9 strains without deletion to CH-FJND-2011 (JQ282909) is 96.6%-99.1%, which is homologous to the nucleotide of vaccine strain DR13 (EU054930) The sex is 95.8%-97.1%. Phylogenetic tree analysis showed that the 9 strains are closely related to the CH-FJND-2011 strain currently circulating in my country, and the Chinese isolate CH/S (JN547228) and the Korean isolate DR13 (JQ023161) are closely related, and they are in the same branch. SD-5 and HEB-1 are closely related to my country's attenuated vaccine strain CV777 (GU372744) and Korean attenuated vaccine strain DR13 (EU054930) and DBI8659 (GU937797) respectively (see figure 2 ). figure 2 In, "▲" is the sequence of the strain obtained in the present invention.

[0046] Example 2 Optimization of RT-PCR reaction conditions

[0047] (1) Differential diagnosis RT-PCR amplification and determination of the best Tm value

[0048] The cDNAs of HLJ-1 strain and ZJCZ4 strain were amplified by PCR with identification primer ORF3-JD1/JD2. The total volume of the PCR reaction is 20μL, containing 2μL of 10×PCR buffer, 1.5μL of dNTP (2.5mmol/L), 0.5μL of upstream and downstream primers, 0.5μL of rTaq DNA polymerase, 2μL of cDNA, and add water to 20μL. The PCR program is: 95°C pre-denaturation 5min; 94°C denaturation 1min, annealing for 30s, 72°C extension for 30s, a total of 35 cycles; finally 72°C extension for 10min. Among them, there are 5 gradients of PCR annealing temperature, namely: 45℃, 48℃, 50℃, 52℃, 55℃. The obtained PCR products were observed by 1% agarose gel electrophoresis.

[0049] (2) Determination of the optimal primer concentration for RT-PCR

[0050] On the basis of the above reaction conditions, the upstream and downstream identification primer concentrations were divided into 5 different concentrations: 25mmol/L, 5mmol/L, 2.5mmol/L, 0.5mmol/L, 0.25mmol/L, etc. for RT-PCR amplification. The PCR products were observed by 1% agarose gel electrophoresis.

[0051] (3) Results:

[0052] Using ZJCZ4 strain and HLJ-1 strain as templates, the reaction conditions such as RT-PCR Tm value and primer concentration were optimized. The results showed that when the differential diagnosis RT-PCR reaction system is 20 μL: template cDNA 2 μL, 10×PCR buffer 2 μL, dNTP (2.5mmol/L) 1.5μL, upstream and downstream primers 25mmol/L each 0.5μL, rTaq DNA polymerase 0.5μL, ddH 2 O 13μL; the reaction conditions are: 95℃/5min, 94℃/1min, 50℃/30s, 72℃/30s, 35 cycles; the last 72℃/10min (see image 3 , Figure 4 ). image 3 Medium, M: Maker DL2000; 1-6: use ZJCZ4 strain as template, in sequence 45℃, 48℃, 50℃, 52℃, 55℃, 58℃; 7-12: use HLJ-1 strain as template, sequence It is 45℃, 48℃, 50℃, 52℃, 55℃, 58℃; 13: negative control. Figure 4 Among them, M: Maker DL2000; 1-5 uses ZJCZ4 strain as template; 25, 5, 2.5, 0.5, 0.25 mmol/L in sequence; 6-10 uses HLJ-1 strain as template; in sequence 25, 5, 2.5, 0.5, 0.25mmol/L; 11: negative control.

[0053] Example 3 RT-PCR specific identification test

[0054] Extract the RNA of TGEV, PoRV, PRRSV (HuN4 strain), CSFV and PEDV (HLJ-1 strain and ZJCZ4 strain) stored in our laboratory, and use ORF3-JD1/JD2 primers to verify the specificity of RT-PCR amplification .

[0055] The results showed that only ZJCZ4 and HLJ-1 strains can amplify specific bands of different sizes, about 300 bp and 250 bp in size, respectively, and no specific bands appeared with other viral nucleic acids as templates (see Figure 5 ). Figure 5 Among them, M: Maker DL2000; 1: TGEV; 2: PoRV; 3: PRRSV (HuN4 strain); 4: CSFV; 5: HLJ-1 strain; 6: ZJCZ4 strain; 7: negative control.