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Universal type porcine deltacoronavirus nested RT-PCR detection method

A technology of RT-PCR and coronavirus, which is applied in the direction of microbe-based methods, biochemical equipment and methods, microbiological measurement/testing, etc., can solve the problem of low sensitivity of RT-PCR, and achieve strong specificity and practicability Strong, reliability-enhancing effect

Pending Publication Date: 2019-03-29
YANGTZE UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low sensitivity of ordinary RT-PCR, this patent establishes a nested RT-PCR method for detecting PDCoV on the basis of conventional RT-PCR to improve the sensitivity and specificity of detection

Method used

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  • Universal type porcine deltacoronavirus nested RT-PCR detection method
  • Universal type porcine deltacoronavirus nested RT-PCR detection method
  • Universal type porcine deltacoronavirus nested RT-PCR detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Establishment of porcine D-coronavirus nested RT-PCR detection method

[0030] (1) Design of porcine D-coronavirus nested RT-PCR primers

[0031] Design two pairs of specific primers according to the base sequence in the porcine D-coronavirus N gene, the sequences of the two pairs of primers are respectively:

[0032] PDCoV-O-F: 5'-CAGGTCCCAGAGGAAATCTTA-3' (SEQ ID NO.1)

[0033]PDCoV-O-R: 5'-TTTGGTAGGTGGCTCATAGGT-3' (SEQ ID NO.2);

[0034] PDCoV-I-F: 5'-GAAGACCTCAGGAGCGTGGAA-3' (SEQ ID NO.3);

[0035] PDCoV-I-R: 5'-TGAGTAGGAGAAGGTAAGGGTGA-3' (SEQ ID NO. 4).

[0036] (2) Sampling and RNA extraction

[0037] A: Sample collection: Collect sample serum, centrifuge at 3000r / min for 20 minutes, take 200μL to extract RNA with Trizol RNA extraction solution, and measure the concentration for later use.

[0038] B: Tissue sample processing: Take the liver and lung of the pig samples to be tested, shred them, extract RNA with Trizol RNA extraction solution, and me...

Embodiment 2

[0044] Example 2: Construction of recombinant positive plasmid pMD18-T-N

[0045] (1) Cloning of N gene

[0046] Genomic RNA of porcine D-coronavirus (PEDV) was extracted, using the genomic RNA as a template, using nested RT-PCR outer primers PDCoV-O-F and PDCoV-O-R to amplify part of the conserved sequence of the N gene. The PCR reaction system (25 μl) is: 12.5 μl of 2×1 Step Buffer, 1 μl of PrimeScript 1Step Enzyme Mix, 1 μl each of 20 μmol / L PDCoV-O-F and 20 μmol / L PDCoV-O-R, 1 μl of RNA template, and RNase Free dH 2 O to 25 μl; PCR reaction program: reverse transcription at 50°C for 30 min, pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, 32 cycles, and extension at 72°C for 6 min. % of the agarose gel for electrophoresis identification, the results are as follows figure 1 As shown, a specific DNA band of about 665 bp was obtained, which was consistent with the size of the target DNA fragment.

[...

Embodiment 3

[0053] Embodiment 3: Nested RT-PCR primer characteristic detection

[0054] (1) Specificity test: extract porcine reproductive and respiratory syndrome (PRRSV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), Escherichia coli and golden yellow The Staphylococcus genome was used as a template to detect the specificity of the two pairs of primers PDCoV-O-F, PDCoV-O-R, PDCoV-I-F, and PDCoV-I-R used in nested RT-PCR, and the specificity of the two pairs of primers was tested. The results are as follows: figure 1 , as shown in 2, both pairs of primers have good specificity and high amplification efficiency.

[0055] (2) Sensitivity test: The positive plasmid obtained in Example 2 was diluted 10-fold to single-digit copy number, and the plasmids of each dilution were selected as templates for the first PCR with primers PDCoV-O-F and PDCoV-O-R Amplify, PCR product detects with 1% agarose gel, the result is as follows ...

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Abstract

The present invention provides nested RT-PCR primers, a detection method and a kit used for detecting porcine deltacoronavirus. The nested RT-PCR primers are two pairs of designed primers according toa highly conserved specific sequence of the porcine deltacoronavirus. The detection method comprises the following steps: sample RNA is extracted; the obtained sample RNA is used as a template, and the primers are used to conduct an RT-PCR reaction; and a reaction product is subjected to an agarose gel electrophoresis analysis. The kit comprises the primers, an amplification reagent, a positive control and a negative control. A provided technical scheme is strong in specificity, high in sensitivity, good in an anti-interference performance and also strong in reliability, overcomes problems oflow sensitivity, poor specificity, etc. of common detection methods, and is relatively low in costs, short in detection cycles and strong in practicability compared with existing detection methods ofnucleic acid hybridization, gene chips, etc.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis, in particular to a nested RT-PCR detection method for porcine D-coronavirus. Background technique [0002] Porcine deltacoronavirus (Porcine deltacoronavirus, PDCoV) is a member of the genus Coronaviridae (Coronavirinae) deltacoronavirus, and it is a porcine enterovirus that has been discovered in recent years. The clinical symptoms are acute diarrhea, vomiting, and dehydration. The virus was first reported in Hong Kong in 2012. After 2014, the disease was reported in the United States, South Korea and other countries one after another. After that, it broke out and spread around the world, which caused a certain impact on the healthy development of the pig industry. Impact. [0003] Porcine D-coronavirus is a member of Deltacoronavirus (Deltacoronavirus), which belongs to the Coronaviridae subfamily Coronaviridae, and its genome composition and arrangement are similar to other coron...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6848C12N15/11C12R1/93
CPCC12Q1/6848C12Q1/701
Inventor 刘国平常小云晋钱钱曾攀胡利群申瑶
Owner YANGTZE UNIVERSITY
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