Primers and probe for detecting hog cholera virus based on a digital PCR technology, a kit and a method thereof

A swine fever virus and technical detection technology, applied in the field of molecular biology, can solve the problems of absolute quantitative detection of swine fever virus, achieve broad application prospects and industrialization prospects, not easily affected, and accurately detect the effect of virus content

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, the prior art does not use digital PCR technology to carry out relevant research on the absolute quantitative detection of swine fever virus, whether it is feasible, and the finished kit suitable for industrialization.

Method used

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  • Primers and probe for detecting hog cholera virus based on a digital PCR technology, a kit and a method thereof
  • Primers and probe for detecting hog cholera virus based on a digital PCR technology, a kit and a method thereof
  • Primers and probe for detecting hog cholera virus based on a digital PCR technology, a kit and a method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044]Embodiment 1 is based on the establishment of the kit for the absolute quantitative detection of swine fever virus by digital PCR technology

[0045] A kit for the absolute quantitative detection of classical swine fever 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 classical swine fever 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 the cell culture of classical swine fever virus, and the negative control is deio...

Embodiment 2

[0053] Embodiment 2 Absolute Quantitative Detection Method Based on Digital PCR Technology to Detect Classical Swine Fever Virus

[0054] The method utilizing the test kit of embodiment 1 to detect classical swine fever 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 2 minutes ...

Embodiment 3

[0079] Example 3 specificity verification

[0080] The serum samples of healthy pigs are detected respectively with the kit of the present invention, and clinically obtained pseudorabies virus, porcine respiratory and reproductive syndrome American type virus, porcine epidemic diarrhea virus, porcine circovirus, Haemophilus parasuis, Streptococcus suis, There are a total of 8 samples of Actinobacillus pleuropneumoniae samples, all samples have been verified by sequencing method, and the test results can be found in figure 1 .

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PUM

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Abstract

The invention discloses a pair of primers and a probe for detecting hog cholera virus based on a digital PCR technology, a kit and a method thereof. The primers and the probe include an upstream 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 PCR technique, judging whether the sample to be tested contains hog cholera 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 ofswine fever, 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 swine fever virus based on digital PCR technology. Background technique [0002] Classical swine fever (CSF; also known as Hog cholera, HC) is an acute, febrile, contagious infectious disease caused by classical swine fever virus (CSFV). Membranes, mucous membranes, and internal organs have varying degrees of bleeding, and swelling of lymph nodes throughout the body, etc., which seriously threatens the development of the pig industry and has brought huge losses to the pig industry. sick. CSFV belongs to the Pestivirus genus of the family Flaviviridae. The genome is a single-stranded positive-sense RNA with a size of about 12.5kb, including an open reading frame, a highly conserved 5'-terminus non-coding region and a 3'-terminus non-coding region. [0003] The current methods for detecting CSFV mainly include traditiona...

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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