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Porcine reproductive and respiratory syndrome virus European type, American type double droplet digital PCR absolute quantitative detection kit

A technology for respiratory syndrome and pig reproduction, which is applied in the measurement/inspection of microorganisms, microorganisms, recombinant DNA technology, etc. It can solve the problem of inability to accurately quantify viral nucleic acids, unable to very well reflect the core concept of digital PCR, and long time required for the test. And other issues

Active Publication Date: 2020-02-21
CHINA ANIMAL DISEASE CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional PRRSV detection methods have disadvantages such as the need to use high-cost instruments and equipment, and the test takes a long time
Although RT-PCR and fluorescent RT-PCR have the characteristics of stronger specificity, high sensitivity, good repeatability, and high degree of automation compared with previous methods, the above methods can only achieve qualitative and semi-quantitative detection, and cannot detect viral nucleic acids. For accurate quantification, there are still some limitations in sensitivity and sensitivity specificity
[0004] The concept of digital PCR (Digital PCR, dPCR) was adopted and published by Bert Vogelstein as early as 1999, and its original intention was to obtain a large number of normal somatic cells from clinical samples (such as urine, lymph, plasma, stool, etc.) A small amount of mutant cells were detected in the sample, but because the consumables that could be used to dilute samples at that time were only 384-well plates, it could not reflect the core concept of digital PCR - "terminal dilution" (terminal dilution) very well.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1, design and screening of primers and probes

[0074] 1. Design and screening of primers and probes for detection of European strains of porcine reproductive and respiratory syndrome virus

[0075] Through a large number of sequence acquisition, analysis, comparison and preliminary experiments, four primers and two probes were designed and preliminarily screened. The nucleotide sequences are as follows:

[0076] LV-F1 (sequence 1 of the sequence listing): 5'-GCTGCMGGTTGCYCATACA-3';

[0077] LV-R1 (sequence 2 of the sequence listing): 5'-CAATCGHGGCCATTCAC-3';

[0078] LV-P1 (SEQ ID NO: 3 of the Sequence Listing): 5'-TGCGBCTGATTCGCGTDACTTC-3';.

[0079] LV-F2: 5'-TGCTGCMGGTTGCTCATAC-3';

[0080] LV-R2: 5'-TCAATCGCGKCCATTCA-3';

[0081] LV-P2: 5'-CGCMTGATTCGCGTGACTTCTAVATCC-3'.

[0082] In the above nucleotide sequence, M stands for A or C, Y stands for C or T, H stands for A, T or C, B stands for G, T or C, D stands for G, A or T, M stands for A or C, K ...

Embodiment 2

[0108] Embodiment 2, optimization of relevant reaction parameters in digital PCR

[0109] 1. Optimization of annealing temperature

[0110] 1. Take the European strain of porcine reproductive and respiratory syndrome virus and extract total RNA.

[0111] 2. Take the American strain of porcine reproductive and respiratory syndrome virus, and extract the total RNA.

[0112] 3. Mix the total RNA obtained in step 1 with the total RNA obtained in step 2 to obtain mixed RNA.

[0113] 4. Using the mixed RNA obtained in step 3 as a template, carry out digital RT-PCR with a primer combination consisting of primer probe set LV-F1R1P1 and primer probe set VR2332-F1R1P1.

[0114] (1) Prepare the following system (20μL): 10μL 2×One-step RT-ddPCR Supermix for probes, 1μL LV-F1, 1μL LV-R1, 0.5μL LV-P1, 1μL VR2332-F1, 1μL VR2332-R1, 0.5 μL VR2332-P1, 2 μL template, 3 μL RNase Free dH 2 O. In the system, the concentration of the upstream primer is 0.5 μM, the concentration of the downstre...

Embodiment 3

[0140] Embodiment 3, the preparation of kit

[0141] 1. Preparation of each reagent

[0142] Solution A is the one-step ddPCR probe master mix. The composition of each 900 μL solution A is as follows: 500 μL 2×One-step RT-ddPCR Supermix for probes, 90 μL LV-F1 solution (the concentration of LV-F1 in the LV-F1 solution is 10 μM), 90 μL VR2332-F1 solution (VR2332-F1 The concentration of VR2332-F1 in the solution is 10 μM), 90 μL of LV-R1 solution (the concentration of LV-R1 in the LV-R1 solution is 10 μM), 90 μL of VR2332-R1 solution (the concentration of VR2332-R1 in the VR2332-R1 solution is 10 μM) , 20 μL LV-P1 solution (the concentration of LV-P1 in the LV-P1 solution is 10 μM), 20 μL VR2332-P1 solution (the concentration of VR2332-P1 in the VR2332-P1 solution is 10 μM).

[0143] Solution B is droplet generating oil.

[0144] Solution C is a positive control. The preparation method of solution C: extract the total RNA of the European type of porcine reproductive and resp...

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PUM

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Abstract

The invention discloses a double droplet digital PCR absolute quantitative detection kit for European and American porcine reproductive and respiratory syndrome virus. The primer probe combination comprises LV-F1R1P1 and VR2332-F1R1P1, wherein LV-F1R1P1 consists of LV-F1, LV-R1 and LV-P1, the LV-F1 is as shown in a sequence 1, LV-R1 is as shown in a sequence 2, and LV-P1 is as shown in a sequence 3; VR2332-F1R1P1 consists of VR2332-F1, VR2332-R1 and VR2332-P1, VR2332-F1 is as shown in a sequence 4, VR2332-R1 is as shown in a sequence 5, and VR2332-P1 is as shown in a sequence 6. The kit has an important application value on prevention and control of European and / or American porcine reproductive and respiratory syndrome viruses, and is favorable to epidemic situation control from the source and effective prevention of porcine epidemic large-scale outbreak.

Description

technical field [0001] The invention belongs to the field of virus detection, and in particular relates to a kit for absolute quantitative detection of porcine reproductive and respiratory syndrome virus European type and American type double droplet digital PCR. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). Characteristic highly contagious infectious disease. The disease broke out in the United States for the first time in 1987, and then appeared in Canada, Europe and Asia one after another. At present, it has become popular all over the world, causing huge economic losses every year. PRRS first appeared in my country in 1995, and then spread rapidly. In 2006, a pig "high fever disease" with high body temperature, high morbidity and high mortality broke out in my c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2600/166C12Q2521/107C12Q2563/159C12Q2563/107C12Q2531/113
Inventor 原霖宋晓晖王传彬周智汪葆玥杨林吴佳俊倪建强韩焘訾占超王晓英毕一鸣王静陈亚娜
Owner CHINA ANIMAL DISEASE CONTROL CENT
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