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TaqMan fluorescent quantitative RT-PCR detection kit for parainfluenza virus 5 (PIV5)

A detection kit and fluorescence quantitative technology, which can be used in the determination/inspection of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of time-consuming and laborious, poor specificity and sensitivity, and high cost

Pending Publication Date: 2021-05-18
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the shortcomings of the existing PIV5 detection methods such as time-consuming and laborious, poor specificity and sensitivity, and high cost, and provide a one-step fluorescent quantitative RT-PCR detection that is specific, sensitive, fast, simple, and can quantitatively detect PIV5 Kit, used in the detection of PIV5 and the quality control of veterinary biological products

Method used

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  • TaqMan fluorescent quantitative RT-PCR detection kit for parainfluenza virus 5 (PIV5)
  • TaqMan fluorescent quantitative RT-PCR detection kit for parainfluenza virus 5 (PIV5)
  • TaqMan fluorescent quantitative RT-PCR detection kit for parainfluenza virus 5 (PIV5)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1——Parainfluenza virus type 5 TaqMan fluorescent quantitative RT-PCR detection kit for detection of veterinary biological products, virus seeds and raw and auxiliary materials

[0064] 1. Sample Processing

[0065] (1) Sample type: Highly pathogenic porcine reproductive and respiratory syndrome live vaccine (TJM-F92 strain), porcine reproductive and respiratory syndrome live vaccine (CH-1R strain), pseudorabies live vaccine (Bartha-K61 strain) , goat pox live vaccine (AV41 strain), Peste des Petits Ruminants live vaccine (Clone9 strain), swine fever virus live vaccine (rabbit source), swine fever live vaccine (cell source), chicken Newcastle disease live vaccine (LaSota strain), chicken infectivity Bronchitis live vaccine (H120 strain), duck plague live vaccine, chicken infectious bursal disease live vaccine (NF8 strain), chicken infectious bursal disease live vaccine (D-22 strain), chicken Marek's disease turkey Herpes virus live vaccine (FC-126 strain), chick...

Embodiment 2

[0068] Example 2——Determination of the minimum detection limit of parainfluenza virus type 5 TaqMan fluorescent quantitative RT-PCR detection kit

[0069] (1) Divide the positive standard from 1×10 9 copies / uL to 1×10 1 The 10-fold serial dilution of copies / uL was used as a template, and the established TaqMan fluorescent quantitative RT-PCR detection method for parainfluenza virus type 5 was used for detection. The minimum detection limit of the invented kit was 10 copies / uL (results shown in Figure 1A).

[0070] (2) The PIV5 / 01 strain and the PIV5 / 02 strain that have determined the virus content are respectively diluted 10 times serially as a template, and are detected with the established parainfluenza virus type 5 TaqMan fluorescence quantitative RT-PCR detection method, and the established method Its sensitivity can reach 0.8TCID 50 (results see image 3 ), with good sensitivity.

Embodiment 3

[0071] Example 3—Repeatability detection of parainfluenza virus type 5 TaqMan fluorescent quantitative RT-PCR detection kit

[0072] Take 1×10 8 copies / uL to 10 6 The positive standard diluted in copies / uL was used as a template, and the samples of each concentration were repeated three times, and the coefficient of variation was calculated. To analyze the reproducibility of the established fluorescent quantitative RT-PCR detection method. The results are shown in Table 1. The coefficients of variation of different template concentrations are all less than 1.5%, indicating that the established fluorescent quantitative RT-PCR detection method has good stability.

[0073] Table 2 PIV5 TaqMan fluorescence quantitative RT-PCR detection method repeatability detection

[0074]

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Abstract

The invention relates to a TaqMan fluorescent quantitative RT-PCR detection kit for a parainfluenza virus type 5 (PIV5). The kit comprises a pair of specific primers and a TaqMan probe which are used for detecting the PIV5. The kit disclosed by the invention is good in detection result specificity and good in PIV5 amplification curve, and other veterinary virus biological products and raw and auxiliary materials do not have specific amplification curves; the sensitivity is high, and the detection limit is 10 copies / mu L when a positive standard substance diluted by 10 times of gradient is detected; the kit is rapid, simple and convenient to detect, reverse transcription and PCR are carried out in one reaction tube, and pollution opportunities are reduced; and the TaqMan fluorescent quantitative RT-PCR detection kit for the PIV5 solves the problems of long time consumption, high labor consumption, poor specificity and sensitivity, high cost and the like of an existing PIV5 detection method, can be used for pollution detection of the PIV5 in the veterinary biological products and the raw and auxiliary materials, and has important significance for detection of the PIV5, quality control of the veterinary biological products and the like.

Description

technical field [0001] The invention relates to a TaqMan fluorescent quantitative RT-PCR detection kit for parainfluenza virus type 5, which belongs to the field of biotechnology. Background technique [0002] Parainfluenza virus type 5 (Parainfluenza virus 5, PIV5) is a single-strand negative-sense non-segmented RNA virus, belonging to Paramyxoviridae (Paramyxoviridae), Paramyxovirinae (Paramyxovirinae), mumps virus (Rubula- virus), which can cause respiratory infections in dogs, cats, pigs, cattle, hamsters, guinea pigs and other animals and humans. The diameter of PIV5 virions is 150-200nm, usually round. PIV5 can infect a variety of cells including human cells, and high-titer virus can be obtained in most cells, among which African green monkey kidney cells (Vero cells) are more sensitive. PIV5 is unstable to heat and is easily destroyed in acid-base solution. The virus has hemagglutination, and can agglutinate red blood cells including guinea pigs, dogs, rabbits, chi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2563/107C12Q2545/114C12Q2561/101C12Q2521/107
Inventor 吴华伟刘丹秦义娴陈晓春高金源孔冬妮高月异黄小洁
Owner CHINA INST OF VETERINARY DRUG CONTROL