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A detection kit capable of simultaneously detecting and differentiating foot-and-mouth disease and vesicular stomatitis, as well as primers and probes

A technology of vesicular stomatitis and a kit, which is applied in the field of inspection and quarantine, can solve the problems of limited application, difficulty in judging results, and low sensitivity, and achieve specificity and exclusivity, saving detection time and cost, coverage and versatility Good results

Active Publication Date: 2020-12-25
中国海关科学技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the currently established methods are single-target detection for one pathogen. In the actual detection, it needs to be repeated many times to complete the purpose of detecting different pathogens. The detection workload is large and the time is long, which cannot meet the rapid clearance of large-scale animal quarantine. the actual needs of
And it is difficult to realize the differential diagnosis of mixed infections and diseases with similar symptoms in actual samples
While establishing single-target pathogen detection technology, veterinary institutions in various countries have also developed multiplex PCR that can detect multiple viruses at the same time, but the disadvantages of traditional PCR technology, such as low sensitivity and difficult judgment of results, limit its application in multiplex detection

Method used

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  • A detection kit capable of simultaneously detecting and differentiating foot-and-mouth disease and vesicular stomatitis, as well as primers and probes
  • A detection kit capable of simultaneously detecting and differentiating foot-and-mouth disease and vesicular stomatitis, as well as primers and probes
  • A detection kit capable of simultaneously detecting and differentiating foot-and-mouth disease and vesicular stomatitis, as well as primers and probes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Composition and use of embodiment 1 test kit

[0039] 1. The composition of the kit

[0040] Table 2 Composition of the kit

[0041]

[0042]

[0043] Among them, 2×Multiplex RT-PCR buffer and Multiplex enzymes were purchased from Path-ID TMMultiplex one-step RT-PCR kit from AB Company.

[0044] The primer mix includes 2 forward primers, 3 reverse primers for FMDV, 2 forward primers, 1 reverse primer for VSV-NJ, 1 forward primer and 1 reverse primer for VSV-IND, primers The sequence is shown in Table 1. The working concentration of the mixed primers is 400nM, and the primers are synthesized by Dalian Bao Biology Co., Ltd.

[0045]The FMDV probe mixture includes 2 probes for FMDV, 1 probe for VSV-NJ and 1 probe for VSV-IND. The probe sequences are shown in Table 1, and the concentrations are 200nM. The probes were synthesized by Dalian Bao Biology Co., Ltd.

[0046] The negative control is sterile water without nucleic acid.

[0047] FMDV positive control is in...

Embodiment 2

[0092] Embodiment 2, the sensitivity test of kit

[0093] 1. Materials

[0094] Foot-and-mouth disease virus and vesicular stomatitis virus are kept in our laboratory.

[0095] 2. Method

[0096] 1) Preparation of positive standard

[0097] Method as described in Example 1.

[0098] 2) Quantitative determination of standard

[0099] Take the prepared in vitro transcribed cRNA and make 200-fold dilutions with RNase-free sterilized water, and measure its absorbance values ​​at 260 nm and 280 nm (OD 260 and OD 280 ), calculate the concentration and purity of the sample to be tested. Pure RNA: 1.7260 / OD280 2.0 indicates that there may be residual isothiocyanate). Concentration of RNA sample (μg / μL): OD 260 ×Dilution factor×40 / 1000, and calculate the copy number (Copies / μL) according to the following formula:

[0100]

[0101] Standard curve drawing:

[0102] Perform 10-fold gradient dilution of the FMDV in vitro standard and then perform fluorescent RT-PCR. Use the r...

Embodiment 3

[0115] Embodiment 3, the specificity test of kit

[0116] 1 material

[0117] The viruses used in this experiment are listed in Table 6.

[0118] Table 6 Viruses and nucleic acids used in the specificity test research process

[0119] Virus source Foot-and-mouth disease virus (FMDV) The lab saves Vesicular stomatitis virus Indiana type (VSV-IND) The lab saves New Jersey-type vesicular stomatitis virus (VSV-NJ) The lab saves Bovine Enzootic Leukemia Virus (BVDV) The lab saves Pseudorabies virus The lab saves Bluetongue Virus (BTV) The lab saves transmissible gastroenteritis virus (TGEV) The lab saves

[0120] 2. Method

[0121] 2.1 Use the primers and probes of foot-and-mouth disease virus, New Jersey-type vesicular stomatitis virus and Indiana-type foot-and-mouth disease virus to perform fluorescent RT-PCR detection on the other 6 viral nucleic acids in the table to verify the specificity of the primers and pro...

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Abstract

The invention discloses a detection kit, primer and probe capable of simultaneously detecting and identifying a foot-and-mouth disease and vesicular stomatitis. The primer and the probe are shown as sequence tables SEQ ID NO:1 to SEQID NO:14. The invention further discloses a one-step-method multiplex fluorescence RT-PCR detection kit rapid, accurate and convenient to use and simultaneously detecting and identifying the foot-and-mouth disease and the vesicular stomatitis. By adopting the detection method, one-time sampling and one-time analysis can be achieved, the purpose of simultaneously detecting and distinguishing three types of important viruses can be achieved, the working amount and cost for detection is reduced, epidemic disease detection can be completed within the shortest time, and time is won for disease prevention and treatment.

Description

technical field [0001] The invention relates to a one-step multiplex fluorescent RT-PCR detection reagent capable of simultaneously detecting and distinguishing foot-and-mouth disease virus (FMDV), New Jersey vesicular stomatitis virus (VSV-NJ) and Indian vesicular stomatitis virus (VSV-IND) The box, primers and probes can achieve the purpose of one-time sampling, one-time analysis, and simultaneous detection and distinction of three viruses, belonging to the field of inspection and quarantine. Background technique [0002] Foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV) are viral diseases that cause infection in cattle, sheep, pigs and other animals. The clinical features are blisters and erosions in the oral mucosa, nipple skin and hoof crown skin. There will be mixed infection, the clinical symptoms are similar, and the differential diagnosis is difficult. They are the most serious pathogens that endanger the breeding industry. Foot-and-mouth dis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2521/107C12Q2537/143C12Q2563/107
Inventor 史喜菊刘艳华李炎鑫刘全国马贵平柏亚铎张伟李冰玲高志强槐硕万晓楠张沫琦
Owner 中国海关科学技术研究中心
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