A duplex PCR method for detection of porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus
A porcine epidemic diarrhea and infectious technology, applied in the direction of biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of unsuitable epidemiological investigation, time-consuming and laborious, unsuitable clinical rapid diagnosis, etc., and achieve high sensitivity and detection High efficiency and strong specific effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0049] 1 RNA extraction
[0050] The method is the same as in Comparative Example 1.
[0051] 2 cDNA synthesis
[0052] The method is the same as in Comparative Example 1.
[0053] 3 Establishment of Multiplex PCR
[0054] RNA was extracted from samples suspected of being infected with TGEV or (and) PEDV, and cDNA was obtained by transcribing it. Using this cDNA as a template, two pairs of primers whose sequences were SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 were simultaneously added to the amplification Amplify the system and establish a double PCR amplification reaction system. The double PCR reaction system was as follows: 1 μl of cDNA template, 0.5 μl of each pair of upstream and downstream primers, 12.5 μl of PCRMix, and 50 μl of ultrapure water. PCR reaction conditions: pre-denature at 95°C for 5 minutes, enter a cycle of 95°C for 60 seconds, 52°C for 1 minute, and 72°C for 1 minute, cycle 35 times, and finally extend at 72°C for 5 minutes. After PCR, t...
Embodiment 2
[0057] Example 2 Verifies the specificity experiment of multiplex PCR.
[0058] 1 RNA extraction
[0059] The method is the same as in Comparative Example 1.
[0060] 2 cDNA synthesis
[0061] The method is the same as that in Comparative Example 1. The RNA of porcine respiratory and reproductive syndrome virus (PRRSV), classical swine fever virus (CSFV) and porcine rotavirus (PRAV) is extracted and reverse-transcribed to obtain cDNA, which is carried out with the cDNA templates of PEDV and TGEV. Multiplex RT-PCR reactions.
[0062] 3 Multiplex PCR detection
[0063] Using the cDNA synthesized in step 2 as a template, 2 pairs of primers whose sequences are SEQIDNO.1, SEQIDNO.2 and SEQIDNO.3, SEQIDNO.4 were simultaneously added to the amplification system to establish a double PCR amplification reaction system. The double PCR reaction system was as follows: 1 μl of cDNA template, 0.5 μl of each pair of upstream and downstream primers, 12.5 μl of PCRMix, and 50 μl of ultrapu...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More