Triplet fluorescence quantitative detection kit for PRRSV (Porcine reproductive and respiratory syndrome virus), HP-PRRSV (Highly pathogenic porcine reproductive and respiratory syndrome virus) and CSFV (Classical Swine Fever Virus)

A technology of porcine PRRS virus and detection kit, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, microorganisms, etc., can solve the problems of no simultaneous detection, reduce detection costs, save detection time, and have high sensitivity Effect

Active Publication Date: 2015-07-01
北京联拓创盈科技有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant reagent that can simultaneously detect porcine PRR

Method used

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  • Triplet fluorescence quantitative detection kit for PRRSV (Porcine reproductive and respiratory syndrome virus), HP-PRRSV (Highly pathogenic porcine reproductive and respiratory syndrome virus) and CSFV (Classical Swine Fever Virus)
  • Triplet fluorescence quantitative detection kit for PRRSV (Porcine reproductive and respiratory syndrome virus), HP-PRRSV (Highly pathogenic porcine reproductive and respiratory syndrome virus) and CSFV (Classical Swine Fever Virus)
  • Triplet fluorescence quantitative detection kit for PRRSV (Porcine reproductive and respiratory syndrome virus), HP-PRRSV (Highly pathogenic porcine reproductive and respiratory syndrome virus) and CSFV (Classical Swine Fever Virus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Design and Screening of Primers and Probes

[0036] In RT-PCR detection, the design of primers and probes directly affects the specificity and sensitivity of detection. Especially in multiple RT-PCR detection, there is mutual influence between different primers and probes. After fully considering relevant factors, the present invention designed several sets of primers and probes, but they were not satisfactory. However, it was unexpectedly found that the following sets of primers and probes showed good specificity and sensitivity.

[0037] 1. Specific primers for CSFV

[0038] Upstream primer:TTCAATTTGGTTCAGGGCCTCC

[0039] Downstream primer: TACTCAGGACTTAGACCACCCAGG

[0040] Specific probes for classical swine fever virus

[0041] Fluorescent probe: CY5-ATGCCCATAGTAGGACTAGCAAACGG-BHQ3

[0042] 2. Specific primers for the American strain of PRRS virus

[0043] Upstream primer: TGGTTTCTCTCTGGCTTTTAGGTC

[0044] Downstream primer: GTAGGTTCCATCTGGTGCGGT

...

Embodiment 2

[0053] Example 2 kit assembly

[0054] According to the physical and chemical properties of the reagents, the kit is divided into two parts A and B for assembly. Part A is the virus total RNA extraction reagent, and part B is the fluorescent quantitative PCR detection reagent, which is convenient for storage and transportation.

[0055] Pack the following reagents in suitable outer boxes and label them (name, batch number, production date, expiry date, etc.).

[0056] Part A

[0057] (1) One bottle of solution A (TRIzoL), 25mL;

[0058] (2) One bottle of solution B (chloroform / isoamyl alcohol), 6mL;

[0059] (3) One bottle of solution C (isopropanol), 18mL;

[0060] Part B

[0061] (4) Solution D (RNase Free dH 2 O) two, 2mL;

[0062] (5) One solution E (triple RT-QPCR reaction solution), 1.5mL;

[0063] (6) One solution F (mixed enzyme solution), 65 μL;

[0064] (7) One bottle of solution G (positive control), 40 μL;

[0065] (8) One bottle of solution H (negative c...

Embodiment 3

[0089] Embodiment 3 specificity experiment

[0090] 1. Reagents and materials

[0091] 1.1 Reagent The kit of Example 2 was used.

[0092] 1.2 Test materials

[0093] 1.2.1 Common related samples: porcine healthy somatic cell culture, porcine circovirus, pseudorabies virus, swine influenza virus, porcine parvovirus, porcine rotavirus, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus .

[0094] 1.2.2 Positive samples: tissue and cell culture of porcine PRRS virus (American strain), tissue and cell culture of highly pathogenic porcine PRRS virus, tissue and cell culture of swine fever virus, and the first three mixture.

[0095] Ten copies of each of the above test materials.

[0096] 2. Test method

[0097] Reagent preparation: with absolute ethanol (75mL, add 25mL RNAse Free dH 2 O,) is configured into 75% ethanol, pre-cooled before use.

[0098] 2.1 Total RNA extraction

[0099]2.1.1 Take 100 μL of tissue sample grinding supernatant and pu...

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Abstract

The invention provides a triplet fluorescence quantitative detection kit for PRRSV (Porcine reproductive and respiratory syndrome virus), HP-PRRSV (Highly pathogenic porcine reproductive and respiratory syndrome virus) and CSFV (Classical Swine Fever Virus). The triplet fluorescence quantitative detection kit comprises primers and probes listed in SEQ ID No.1-9 in a sequence table, as well as a total virus RNA extraction agent, an RT-QPCR mixed enzyme solution, RNase Free dH2O, and one or more of positive controls and negative controls. The kit is based on a triplet fluorescence quantitative PCR technology, PRRSV molecules, HP-PRRSV molecules and CSFV molecules are simultaneously detected, the kit is applied to detection of insignificant-content samples such as animal lungs, lymph glands, spleens, kidneys, hearts, livers, blood and meat products, whether the sample donors are contaminated or not is determined, and the kit is applicable to quarantine of live animals and meat products. The kit has the advantages of good specificity and high sensitivity, the detection cost is greatly lowered through joint detection, the detection time is saved, and the type of a pestilence can be conveniently determined in a short time at an epidemic outbreak.

Description

technical field [0001] The invention relates to the field of virus diagnosis, in particular to a triple fluorescent quantitative RT-PCR detection kit capable of simultaneously detecting porcine blue-ear disease virus, highly pathogenic porcine blue-ear disease virus and swine fever virus. Background technique [0002] Classical swine fever is a highly contagious febrile infectious disease caused by classical swine fever virus (CSFV), which is highly contagious and highly pathogenic. Pigs are the only natural host of the disease. The main manifestations are characteristic pathological changes, visceral hemorrhage, infarction and necrosis, and the mortality rate is very high, causing significant economic losses to the pig industry. Sick pigs are the main source of infection, and direct contact between susceptible pigs and sick pigs is the main way of virus transmission. In my country, the epidemic of swine fever presents the coexistence of typical swine fever and atypical swi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 陈晨冯小宇宋彦军韦海涛周德刚刘晓东马付坤闫慧张静依
Owner 北京联拓创盈科技有限公司
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