SN/T 4675.29-2016 in English

VALID

Determinetion of Brettanomyces bruxellensis in wine for export―Real-time PCR method

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

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Standard No:	SN/T 4675.29-2016
Document status:	VALID
Title in English:	Determinetion of Brettanomyces bruxellensis in wine for export―Real-time PCR method
Title in Chinese:	出口葡萄酒中酒香酵母检验实时荧光PCR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
Related Topics:	pcr Fluorescent PCR Multiplex Fluorescent PCR Fluorescence inspection acceptance criteria Multiplex fluorescent real-time quantitative PCR Real-time PCR and multiplex PCR Multiplex real-time fluorescent quantitative PCR flow fluorescent pcr Fluorescent PCR+ Fluorescence real-time quantitative multiplex fluorescence multiplex pcr vs fluorescent pcr Isothermal fluorescent PCR relative fluorescence pcr multiplex real-time fluorescent quantitative pcr When the cod experiment is 1 times Multiplex real-time fluorescent PCR instrument Multiplex PCR and fluorescent PCR glucose fluorescence Multiplex PCR real-time fluorescence quantitative PCR Real-time fluorescence quantitative PCR analysis fluorescent pcr peak Q5 real-time fluorescent quantitative PCR Multiplex fluorescent PCR Real-time fluorescent PCR rna pcr method Real-time quantitative PCR, real-time fluorescence Q3 real-time fluorescent quantitative PCR When detecting fluorescence spectra? ????pcr?? Q7 real-time fluorescent quantitative PCR Real-time fluorescent PCR instrument q5 Q7 fluorescent PCR fluorescent strip test Fluorescent temperature pcr standard q6 fluorescent pcr relative real-time fluorescent quantitative pcr Fluorescent quantitative PCR in inspection and quarantine Fluorescence data the time How long is real-time fluorescent quantitative PCR Real-time fluorescent quantitative PCR analysis Portable real-time fluorescent quantitative PCR Fluorescence real-time quantitative PCR real-time pcr analysis method Wine cesium detection real-time fluorescent quantitative pcr and pcr Fluorescence test before pregnancy Q5 fluorescent PCR Real-time fluorescent quantitative PCR peak Fluorescence PCR instrument usage method Fluorescent PCR cassette multiplex fluorescent PCR method multiplex real-time fluorescence quantitative pcr technology Pyrene fluorescence experiment q7 real-time fluorescent PCR instrument Real-time fluorescent fluorescent glucose flow real-time fluorescence wine yeast wine app pcr fluorescence method wine yeast pcr+ method plate time fluorescence PCR initial fluorescence Real-time fluorescence semi-quantitative PCR q6 real-time fluorescence Cadmium in wine fluorescent pcr iso real-time fluorescent quantitative pcr and pcr Baijiu Isothermal PCR Real-time fluorescent PCR laboratory yeast Fluorescence PCR detection series Fluorescence Polarization Experiment experimental fluorescence pcr fluorescence method Yeast immunofluorescence wine opinions PCR produces multiple strips Fluorescence detection limit Flow cytometry fluorescence detection limit Wine testing reagent tartaric acid determination method Detection method of copper in wine

Introduction

Technical Background of the Standard

This standard, part 29 of the SN/T 4675 series, is specifically developed to address the molecular detection needs of Brettanomyces bruxellensis (brettanomyces) in wine export trade. This microorganism can cause phenolic off-flavors in wine and is a key target for international trade monitoring.

Comparison of Method Principles

Detection Dimensions	Traditional Culture Method	Real-time Fluorescence PCR Method
Detection Cycle	5-7 Days	≤4 Hours
Sensitivity	10² CFU/mL	10⁰-10¹ CFU/mL
Specificity	Susceptible to Interference from Miscellaneous Bacteria	Targeting RAD4 Gene

Key Operation Points

DNA Extraction Optimization

PVPP (cross-linked polyvinyl pyrrolidone) combined with liquid nitrogen grinding was used to break the yeast cell wall, and DNA quality was ensured through a two-step purification using CTAB-chloroform (A260/A280 required to be 1.7-1.9).

Primer and probe design

Designed for the specific gene RAD4 of Brettanomyces bruxellensis:
Forward primer: 5'-GTT CAC ACA ATC CCC TCG ATC AAC-3'
Reverse primer: 5'-TGC CAA CTG CCG AAT GTT CTC-3'
FAM-labeled probe: 5'-ATG GCG AGG ATG AAA GTT TGG GAT ACA-3'

Implementation recommendations

The laboratory needs to verify the primer specificity using the NBIRC 0628 standard strain
It is recommended to set up three parallel controls for each batch of testing: a positive control (NBRC 0628), a negative control (TE buffer), and an extraction blank
Critical value determination (Ct value 35-40) requires re-testing and confirmation

Technology Evolution Analysis

Compared to the 2010 version of the culture method, this standard introduces:
1. Magnetic bead purification technology replaces phenol chloride extraction
2. Optimized amplification procedure: 95°C 10 min pre-denaturation → 50 cycles (95°C 30 s → 58°C 20 s → 72°C 20 s)
3. ΔCt value is added to assist analysis of results

More samples: GB 18306-2015 NB/T 47013.2-2015 YD/T 3934-2021

Sample only — not a preview of SN/T 4675.29-2016

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