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Method for detecting influenza virus titer through fluorescent quantitative PCR

An influenza virus, fluorescence quantification technology, applied in microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of long detection time, poor practicability, poor repeatability of results, etc. , High practicability and high repeatability

Inactive Publication Date: 2021-11-09
JIANGSU JINDIKE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] According to the 2020 edition of the Chinese Pharmacopoeia, the virus titer of influenza virus is detected by the chicken embryo EID50 method; however, this method takes a long time to detect, and generally takes 3-4 days for a detection, and the repeatability of the results is poor, usually fluctuating in the range of ±1.0, for the key data, multiple determinations are required to determine the titer, which is less practical

Method used

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  • Method for detecting influenza virus titer through fluorescent quantitative PCR
  • Method for detecting influenza virus titer through fluorescent quantitative PCR
  • Method for detecting influenza virus titer through fluorescent quantitative PCR

Examples

Experimental program
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Embodiment 1

[0037] A method for detecting influenza virus titer by fluorescent quantitative PCR, comprising the following steps:

[0038] (1) Extract the RNA of the sample to be tested:

[0039] ①Take the virus sample, add the lysate, mix well and place it at room temperature for 5 minutes, then add the buffer solution to the lysed sample solution, mix well and transfer the mixture to an affinity column with a liquid connection tube, 13000r / min Centrifuge for 30 seconds and discard the filtrate;

[0040] ② Add 500 μl of washing solution to the affinity column, centrifuge at 13000r / min for 30 seconds, and discard the filtrate;

[0041] ③Repeat step ②;

[0042] ④Put the affinity column into the liquid tube, centrifuge at 13000r / min for 2 minutes, discard the liquid tube and filtrate; then put the affinity column into a new centrifuge tube, and absorb the membrane to the bottom of the affinity column. Add 50 μl of eluent to the center, place at room temperature for 2 minutes, and centrifu...

Embodiment 2

[0057] A method for detecting influenza virus titer by fluorescent quantitative PCR, comprising the following steps:

[0058] (1) Extract the RNA of the sample to be tested:

[0059] ①Take the virus sample, add the lysate, mix well and place it at room temperature for 5 minutes, then add buffer solution to the lysed sample solution, mix well, transfer the mixture to an affinity column with a liquid tube, 13000r / min Centrifuge for 30 seconds and discard the filtrate;

[0060] ② Add 500 μl of washing solution to the affinity column, centrifuge at 13000r / min for 30 seconds, and discard the filtrate;

[0061] ③Repeat step ②;

[0062] ④ Put the affinity column into the liquid tube, centrifuge at 13000r / min for 2 minutes, discard the liquid tube and the filtrate; then put the affinity column into a new centrifuge tube, and absorb the membrane to the bottom of the affinity column. Add 50 μl of eluent to the center, place at room temperature for 2 minutes, and centrifuge at 13,000 ...

Embodiment 3

[0076] A method for detecting influenza virus titer by fluorescent quantitative PCR, comprising the following steps:

[0077] (1) Extract the RNA of the sample to be tested:

[0078] ①Take the virus sample, add the lysate, mix well and place it at room temperature for 5 minutes, then add buffer solution to the lysed sample solution, mix well, transfer the mixture to an affinity column with a liquid tube, 13000r / min Centrifuge for 30 seconds and discard the filtrate;

[0079] ② Add 500 μl of washing solution to the affinity column, centrifuge at 13000r / min for 30 seconds, and discard the filtrate;

[0080] ③Repeat step ②;

[0081] ④ Put the affinity column into the liquid tube, centrifuge at 13000r / min for 2 minutes, discard the liquid tube and the filtrate; then put the affinity column into a new centrifuge tube, and absorb the membrane to the bottom of the affinity column. Add 50 μl of eluent to the center, place at room temperature for 2 minutes, and centrifuge at 13,000 ...

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Abstract

The invention discloses a method for detecting influenza virus titer through fluorescent quantitative PCR. In the prior art, a chick embryo EID50 method is used for detecting the virus titer of influenza virus; however, the method is long in detection time, generally, the detection time is 3-4 days for one time, due to the reasons such as sensitivity of the method, the result repeatability is poor, the fluctuation range is generally + / -1.0, the titer of key data needs to be determined for multiple times, and the practicability is poor. The invention discloses the method for detecting the influenza virus titer through the fluorescent quantitative PCR. The detection time of the method only needs 4-5 hours, the result repeatability is good, the fluctuation of the detection range is controlled within 0.5, and the lowest detection limit is 10 copies. The invention establishes a more convenient and efficient virus titer detection replacement method, the problems that the EID50 method is long in detection time consumption, large in result fluctuation, poor in repeatability and the like are effectively solved, the PCR method is shorter in time consumption, more accurate and efficient, more suitable for detecting the influenza virus titer and higher in practicability.

Description

technical field [0001] The invention relates to the technical field of virus titer detection, in particular to a method for detecting influenza virus titer by fluorescent quantitative PCR. Background technique [0002] Influenza is called influenza for short, and is an acute respiratory infection caused by influenza virus; influenza virus (influenzavirus) is a representative species of Orthomyxoviridae (Orthomyxoviridae), referred to as influenza virus. There are four types of influenza viruses: Type A, Type B, Type C and Type D, and those that have an impact on humans are mainly Type A, Type B and Type C. Among them, influenza A (type A) virus is the only influenza virus known to cause a global influenza pandemic with strong transmissibility; influenza B virus has only one subtype, and has strong host specificity. So far, it has only been found to infect humans and seal. [0003] Influenza C (type C) infection usually causes mild illness and does not lead to a human influ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 杨骏宇朱实惠袁璐李卉茹余军杨文彬吴建华
Owner JIANGSU JINDIKE BIOTECHNOLOGY CO LTD
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