GB/T 35806-2018 in English

VALID

Animal influenza detection—Protocol of duplex real-time RT-PCR for influenza virus subtypes H7 and N9

Issued on:2018-02-06
Implemented on:2018-09-01
File Format:PDF
Delivery:Via email within 1~3 business days

Price(USD)： $160.00
$156.00

Standard No:	GB/T 35806-2018
Document status:	VALID
Title in English:	Animal influenza detection—Protocol of duplex real-time RT-PCR for influenza virus subtypes H7 and N9
Title in Chinese:	动物流感检测 H7N9亚型流感病毒双重荧光RT-PCR检测方法
Language:	English
File Format:	Electronic (PDF)
Delivery:	Via email within 1~3 business days
Issued on:	2018-02-06
Implemented on:	2018-09-01
ICS Classification:	11.220-Veterinary medicine
Chinese Classification:	B41-Animal quarantine, veterinary and epidemic prevention
Professional Classification:	GB-National Standard
Related Keywords:	animal influenza virus animal influenza detection protocol influenza virus subtypes h7 positive h7 subtype influenza virus positive n9 subtype influenza virus
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《GB/T 35806-2018动物流感检测 H7N9亚型流感病毒双重荧光RT-PCR检测方法》由TC181（全国动物卫生标准化技术委员会）归口，主管部门为农业农村部。

Introduction

GB/T 35806—2018 Standard Overview

This standard specifies the TaqMan dual fluorescence RT-PCR operation method for simultaneous detection of H7 and N9 subtype animal influenza virus nucleic acids, which is applicable to the rapid detection and screening of H7 and N9 subtype animal influenza virus nucleic acids.

Detection principle and method

Steps	Description	Key data
Sample collection and processing	Including throat swabs, cloacal swabs and nasal swabs, etc., which must be sent to the laboratory within 24 hours after collection.	- Sampling tools need to be sterilized by high pressure - Sample storage temperature: 2°C~8°C (short term) or -70°C (long term)
Nucleic acid extraction	Use TRIzol lysis method to extract RNA to ensure no DNA contamination.	- Use DEPC water treatment - The extracted RNA needs to be used immediately or stored at -70°C
Fluorescence RT-PCR amplification	Use a dual-channel fluorescence PCR instrument to detect FAM and HEX signals respectively.	- Reaction system: 15μL/tube - Cycle parameters: 40 cycles

Reagents and instruments

Reagent description

Fluorescent RT-PCR reaction solution contains: 5×RT buffer, MgCl₂, dNTP and other key components.

Instruments

Fluorescent PCR detector must have multi-channel detection function, and ABI 7500 or similar equipment is recommended.

Sample collection and pretreatment

Strict aseptic operation is required during sampling, and samples must not be cross-contaminated. The specific steps are as follows:

Throat swab: Scrape deep into the larynx and palate cleft 2 to 3 times and rotate.
Cloacal swab: Scrape deep into the cloaca, rotate once and pick up a small amount of feces.
Nose swab: Scrape deep into the nasal cavity 2 to 3 times and rotate.

Result determination and quality control

Ct value (or Cp value) is the key indicator for judging amplification efficiency:

Detection channel	Result determination	Quality control standard
FAM detection channel positive	H7 subtype influenza virus nucleic acid exists in the sample, and the Ct value is ≤30.0.	- No amplification curve for negative control - Ct value of positive control ≤30.0
HEX detection channel positive	N9 subtype influenza virus nucleic acid is present in the sample, and the Ct value is ≤30.0.	- No amplification curve for negative control - Ct value of positive control ≤30.0

Implementation suggestions

To ensure the accuracy of the test, the laboratory needs to:

Strictly follow the standard operating procedures.
Regularly calibrate the instruments.
Establish a quality control system, including the use of positive and negative controls.
Train the test personnel to ensure that they are familiar with the operation of the reagents and instruments.

More samples: NB/T 47013.2-2015 YD/T 3934-2021 JGJ 94-2008

Sample only — not a preview of GB/T 35806-2018

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