One-step triple RT-PCR detection primer for distinguishing CHUV, BCV and DAV and kit thereof

A RT-PCR and CHUV-P1 technology, which is applied in the field of one-step triple RT-PCR detection primers and kits, can solve the problems of increased experimental costs, virus diffusion, biological safety, and long time-consuming SNT, so as to avoid repeated detection and specificity. Good performance and high sensitivity

Active Publication Date: 2020-01-03
YUNNAN ANIMAL SCI & VETERINARY INST
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is only the traditional serum neutralization test (SNT) for PALV serotype identification technology. Although SNT is the gold standard for virus serotype identification, there are certain shortcomings in actual use: (1) SNT takes a long time , It usually takes about 1 week, which is not conducive to the rapid diagnosis of the epidemic; (2) SNT needs to use live virus, and there is a biological safety hazard of virus spread; (3) The preparation of standard positive serum used in SNT requires the use of standard virus multiple times Preparation of immune-susceptible animals is time-consuming and laborious; (4) For samples mixed with multiple serotype strains, when serotype identification is carried out by SNT, there may be cross-reactions between different serotype strains, and at the same time, a certain strain may be missed. (5) SNT needs to prepare basic materials such as 96-well cell culture plate, cell culture medium, fetal bovine serum, etc., which increases the cost of the experiment
However, multiplex PCR detection methods for CHUV, BCV and DAV have not been established in my country, which seriously hinders the research on the epidemiology and etiology of PALV in my country; Corresponding serotype multiplex PCR detection method

Method used

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  • One-step triple RT-PCR detection primer for distinguishing CHUV, BCV and DAV and kit thereof
  • One-step triple RT-PCR detection primer for distinguishing CHUV, BCV and DAV and kit thereof
  • One-step triple RT-PCR detection primer for distinguishing CHUV, BCV and DAV and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1, the establishment of one-step triple RT-PCR detection method

[0072] 1.1 Design and synthesis of primers

[0073] According to the Seg-2 gene sequence of Chinese CHUV, BCV and DAV strains and the sequence of PALV strain published by GenBank, a pair of specific primers CHUV-P1 and CHUV-P2 for CHUV and a pair of specific primers for BCV were designed respectively. BCV-P1 and BCV-P2, a pair of DAV-specific primers DAV-P1 and DAV-P2; the designed three pairs of specific primers (Table 5) were synthesized by Shanghai Invitrogen Company.

[0074] Table 5 Sequence information of primers for CHUV, BCV and DAV serotype-specific one-step triple RT-PCR detection

[0075]

[0076] All primers were sterile ddH 2 O (RNase free) was prepared at a concentration of 20 μM for use.

[0077] 1.2 Extraction of viral RNA

[0078] Use β-propiolactone to inactivate CHUV, BCV, DAV, BTV, EHDV, AKAV, the specific inactivation process is as follows: the final concentration of...

Embodiment 2

[0089] The specificity test of embodiment 2, triple RT-PCR

[0090] Perform the reaction according to the optimal reaction conditions determined in Example 1-step 1.3, for CHUV+BCV+DAV mixed RNA template (1.5 μL+1.5 μL+1.5 μL), CHUV+BCV mixed RNA template (2.25 μL+2.25 μL) , CHUV+DAV mixed RNA template (2.25μL+2.25μL), BCV+DAV mixed RNA template (2.25μL+2.25μL), CHUV, BCV, DAV, BTV, EHDV, AKAV, AHSV single RNA template (4.5μL) Multiplex RT-PCR amplification. After the reaction, 5 μL of the amplified product was electrophoresed on a 15 g / L agarose gel. The results showed that the CHUV, BCV, and DAV triple RT-PCR detection methods established by the present invention can only be specific for CHUV, BCV, and DAV RNA. The amplification results of BTV, EHDV, AKAV, and AHSV RNA are all negative, indicating that the present invention can be used as a method for specific identification of CHUV, BCV, and DAV serotypes (attached figure 2 ).

Embodiment 3

[0091] Example 3, Sensitivity Verification of Primers

[0092] Get the CHUV Seg-2ssRNA (9.5 × 10 11 copy / μL), BCVSeg-2ssRNA (5.0×10 11 copy / μL) and DAV Seg-2ssRNA (6.9×10 11 copy / μL) equal volumes were mixed and then serially diluted 10 times, choose 10 3 ~10 10 The diluted ssRNA was used as a template, and the corresponding nucleic acid copy numbers were 9.5×10 8 Copy / μL~95 copy / μL (CHUV / Seg-2ss RNA), 5.0×10 8 copy / μL~50 copy / μL (BCV / Seg-2ssRNA), 6.9×10 8 Copy / μL~69 copy / μL (DAV / Seg-2ssRNA); perform multiplex RT-PCR amplification according to the optimized RT-PCR reaction system and conditions determined in Example 1-step 1.3. After the reaction, 5 μL of the amplified product was electrophoresed on a 15 g / L agarose gel. The results showed that the CHUV, BCV, and DAV triple RT-PCR detection method established in this study had a minimum detection amount of 950 copies of CHUV and the lowest BCV. The detection amount is 500 copies, and the minimum detection amount of DAV ...

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Abstract

The invention relates to a one-step triple RT-PCR detection primer for distinguishing CHUV, BCV and DAV and kit thereof, belonging to the technical field of veterinary infectious disease detection. The kit includes 3 pairs of primers, respectively CHUV-P1 and CHUV-P2, BCV-P1 and BCV-P2, DAV-P1 and DAV-P2; and also comprises a blank control template, a positive control template and a PCR amplification reagent; the blank control template is RNase Free Water; there are three positive control templates, namely CHUV, BCV and DAV inactivated viruses respectively. By adopting the kit of the invention, the one-step RT-PCR reaction can detect three PALV serotype virus such as CHUV, BCV and DAV at the same time, avoiding repeated detection of conventional PCR, having the advantages of low cost, highefficiency and the like, and being easy to popularize and apply.

Description

technical field [0001] The invention belongs to the technical field of veterinary infectious disease detection, and in particular relates to a one-step triple RT-PCR detection primer and a kit for distinguishing three PALV serotype viruses CHUV, BCV and DAV. Background technique [0002] Palyam serogroup virus (PALV), a member of the Orbivirus genus of the Reoviridae family, is mainly transmitted to ruminants such as cattle and sheep by the blood-sucking insect Culicoides spp. The bites spread. Among them, cattle are the most susceptible to the disease. Infected pregnant cows have miscarriages, premature births and stillbirths, which can cause Hydranencephaly cerebellar hypoplasia syndrome (HCH) in newborn calves. cause serious economic losses. [0003] The PALV genome consists of 10 segments of double-stranded RNA (Seg-1 ~ Seg-10), encoding 7 structural proteins (VP1 ~ VP7) and 4 nonstructural proteins (NS1, NS2, NS3 and NS3a). Among them, the VP2 protein encoded by Seg-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/166C12Q2537/143C12Q2521/107Y02A50/30
Inventor 廖德芳杨恒杨振兴李占鸿李卓然肖雷李华春
Owner YUNNAN ANIMAL SCI & VETERINARY INST
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