SN/T 4708-2016 in English

VALID

Detection method of vibrio cholerae and vibrio mimicus by duplex real-time PCR at frontier ports

Issued on:2016-12-12
Implemented on:2017-07-01
File Format:PDF
Delivery:Via email within 1~3 business days

Price(USD)： $200.00
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Standard No:	SN/T 4708-2016
Document status:	VALID
Title in English:	Detection method of vibrio cholerae and vibrio mimicus by duplex real-time PCR at frontier ports
Title in Chinese:	国境口岸霍乱弧菌和拟态弧菌双重荧光PCR检测方法
Language:	English
File Format:	Electronic (PDF)
Delivery:	Via email within 1~3 business days
Issued on:	2016-12-12
Implemented on:	2017-07-01
Professional Classification:	SN-Import&Export Inspection
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Introduction

Analysis of Standard Technical Framework

Detection Elements

Vibrio cholerae

Vibrio mimics

Technical Commonalities

Target Gene

ompW Gene

vmh Gene

Conserved Gene Detection

Probe Labeling

FAM-BHQ1

HEX-BHQ1

Dual-channel Fluorescence Detection

Sensitivity

≥10²

Multiple primer design: Specific primer sets for the ompW gene of Vibrio cholerae (FAM channel) and the vmh gene of Vibrio mimics (HEX channel)
Reaction system optimization: 20μL system contains 10μL Premix Ex Taq^TM, and the annealing temperature is uniformly set at 60℃
Interpretation criteria: Ct value ≤35 is positive, 36-40 requires retesting, and >40 is considered negative

Key points for biosafety implementation

According to the requirements of GB19489-2008, the entire testing process must be carried out in a biological safety cabinet:

Sample transportation must comply with the provisions of Order No. 45 of the Ministry of Health
Waste disposal must be sterilized by high pressure (121℃, 30min)
Positive controls use inactivated strains or synthetic DNA fragments

Technological Evolution Analysis

Compared with the traditional culture method (which takes 3-5 days), this standard has significant advantages:

Parameters	Traditional method	This standard method	Improvement range
Test cycle	72-120h	4-6h	20-fold
Sensitivity	10³ CFU/mL	10² copies/mL	10-fold
Specificity	85-90%	99%	Significantly improved

Recommendations for standard implementation

Key points for quality control

Each batch of experiments must include positive and negative controls
Calibrate the micropipette regularly (quarterly calibration is recommended)
Store the fluorescent probe in the dark at -20°C

Handling plan for difficult samples

Inhibitor treatment: For samples with a Ct value greater than 35, dilute them at 1:10 for retesting
Water sample concentration: 450mL of water sample needs to be enriched with 50mL of alkaline peptone water
Cross-contamination prevention and control: Use filter tips and operate in separate areas

More samples: QC/T 942-2021 YD/T 3934-2021 GB 18306-2015

Sample only — not a preview of SN/T 4708-2016

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