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Primers and probe for detecting porcine epidemic diarrhea virus based on digital PCR technology, kit and a method thereof

A technology for porcine epidemic diarrhea and technology detection, applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., to achieve broad application prospects and industrialization prospects, high sensitivity, and avoid economic losses

Inactive Publication Date: 2018-12-07
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, there is no relevant research on the absolute quantitative detection of porcine epidemic diarrhea virus using digital PCR technology, as well as a feasible related scheme and a finished kit suitable for industrialization.

Method used

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  • Primers and probe for detecting porcine epidemic diarrhea virus based on digital PCR technology, kit and a method thereof
  • Primers and probe for detecting porcine epidemic diarrhea virus based on digital PCR technology, kit and a method thereof
  • Primers and probe for detecting porcine epidemic diarrhea virus based on digital PCR technology, kit and a method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Establishment of a kit for the absolute quantitative detection of porcine epidemic diarrhea virus based on digital PCR technology

[0045]A kit for the absolute quantitative detection of porcine epidemic diarrhea virus based on digital PCR technology, including a primer set, 2×RT-ddPCR Supermix, RNase-free distilled water, virus total RNA extraction reagent, positive control and negative control.

[0046] (1) Design of primers for digital PCR amplification: primers were designed with the specific conserved sequence of porcine epidemic diarrhea virus as the target gene. The primer sequences are listed in Table 1.

[0047] Table 1 Primer sequence list

[0048]

[0049] (2) The molar ratio of FP primer, BP primer and probe in the primer set is 3:3:2.

[0050] (3) 2×RT-ddPCR Supermix contains: 2×One-step RT-ddPCR Supermix and 25mMManganous acetate.

[0051] (4) The positive control is porcine epidemic diarrhea virus cell culture, and the negative control is ...

Embodiment 2

[0053] Example 2 The detection method of absolute quantitative detection of porcine epidemic diarrhea virus based on digital PCR technology

[0054] The method utilizing the kit of embodiment 1 to detect porcine epidemic diarrhea virus may further comprise the steps:

[0055] (1) Extraction of viral RNA:

[0056] 1) Take the sample to be tested and the positive control, add 600ul lysate A respectively, vortex and mix for 20 seconds, and let stand at room temperature for 10 minutes;

[0057] 2) Transfer the mixture to an adsorption column and centrifuge at 12,000×g for 30-60 seconds;

[0058] 3) Discard the liquid in the collection tube, add 500ul washing solution B to the adsorption column, and centrifuge at 12,000×g for 30-60 seconds;

[0059] 4) Discard the liquid in the collection tube, add 500ul washing solution C to the adsorption column, and centrifuge at 12,000×g for 30-60 seconds;

[0060] 5) Discard the liquid in the collection tube, and centrifuge at 12,000×g for ...

Embodiment 3

[0079] Example 3 specificity verification

[0080] Use the kit of the present invention to detect serum samples of healthy pigs, clinically obtained pseudorabies virus, porcine respiratory and reproductive syndrome American type virus, swine fever virus, porcine circovirus, Haemophilus parasuis, Streptococcus suis, porcine pleura There are a total of 8 samples of Actinobacillus pneumoniae samples, all of which have been verified by sequencing methods, and the test results can be found in figure 1 .

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Abstract

The invention discloses a pair of primers and a probe for detecting porcine epidemic diarrhea virus based on a digital PCR technology, a kit and a method thereof. The primers and the probe include anupstream primer FP, a downstream primer BP and a probe. The detection kit includes a primer set, 2*RT-ddPCR Supermix, RNase-free distilled water, a viral total RNA extraction reagent, a negative control and a positive control. The detection method comprises the following steps: extracting a sample RNA to be tested, quantitatively detecting the sample RNA to be detected by the microdrop digital PCRtechnique, judging whether the sample to be tested contains porcine epidemic diarrhea virus by the copy number obtained by the amplification analysis, and determining the content thereof. The invention has the advantages of accuracy, sensitivity and wide applicability, has no dependency on a standard curve, can realize absolute quantification, can achieve early detection, early treatment and early prevention of diagnosis of porcine epidemic diarrhea, and can effectively control the outbreak of the epidemic.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a primer, a probe, a kit and a method for detecting porcine epidemic diarrhea virus based on digital PCR technology. Background technique [0002] Porcine Epidemic Diarrhea (PED) is an acute, highly contagious, contagious enteric disease caused by Porcine Epidemic Diarrhea Virus (PEDV), and its clinical symptoms are vomiting, diarrhea and Dehydration etc. Pigs of various ages are all susceptible, and the infection incidence rate of suckling piglets, weaned piglets and fattening pigs can reach 100%, and the mortality rate in suckling piglets is very high, which has brought huge losses to the pig industry. PEDV belongs to the genus Coronaviridae of the family Coronaviridae. Its genome is a single-stranded positive-sense RNA with a size of about 28 kb, including 7 open reading frames, 5' non-coding regions and 3' non-coding regions. [0003] The current detection methods f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2521/107C12Q2563/159
Inventor 叶蕾曹炜伟陈洵常彦磊李丽丽石磊
Owner JINAN UNIVERSITY
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