GH/T 1356-2021 in English

VALID

Identification of Loquat Species in Loquat Honey-Real-time PCR Method

Issued on:2021-11-08
Implemented on:2022-01-01
File Format:PDF
Delivery:Via email within 1~3 business days

Price(USD)： $210.00
$204.00

Standard No:	GH/T 1356-2021
Document status:	VALID
Title in English:	Identification of Loquat Species in Loquat Honey-Real-time PCR Method
Title in Chinese:	枇杷蜜植物源成分的检测实时荧光PCR法
Language:	English
File Format:	Electronic (PDF)
Delivery:	Via email within 1~3 business days
Issued on:	2021-11-08
Implemented on:	2022-01-01
Related Keywords:	loquat honey-real-time pcr method introduction technical background pcr pre-amplification technology digital pcr loquat species pcr preparation area loquat honey
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Introduction

Technical background and evolution of standards

GH/T 1356-2021 is my country's first molecular detection standard for plant-derived ingredients in loquat honey, marking the leap from traditional physical and chemical analysis to the genetic level for authenticity identification of bee products. The standard is based on the loquat (Rhaphiolepis bibas L.) chloroplast genome-specific sequence (GenBank ID: kj170769) and significantly improves the recovery rate of degraded DNA in honey by optimizing the CTAB extraction process.

Core Detection Principle

Technical Elements	Parameter Requirements	Technical Advantages
Target Sequence	166bp fragment of chloroplast genome	Species specificity reaches 99.7%
Probe Design	FAM-TAMRA Dual Labeling	Detection Limit as Low as 0.1pg/μL
Ct Value Determination	≤37 is positive	False Negative Rate <0.5%

Key Operation Points

DNA Extraction Optimization Scheme

The standard innovatively adopts the CTAB-sorbitol combined lysis system, among which:

5% sodium lauryl sarcosine can effectively dissociate honey glycoprotein complex
PVP-40 can remove polyphenol interfering substances
-20℃ isopropanol precipitation can improve the recovery rate of trace DNA

Actual cases show that this scheme increases the OD260/280 qualified rate from 62% of the conventional method to 89%.

Implementation recommendations

Laboratory zoning: Strictly distinguish between pre-treatment area, PCR preparation area and amplification area
Negative control setting: It is recommended to add common nectar sources such as rapeseed and acacia
Equipment calibration: Fluorescence PCR instrument needs to be calibrated with ROX fluorescence every month
Method validation: New laboratories should conduct more than 50 reproducible validations

Technical limitations and development

The current method has a detection sensitivity of about 30% for deeply processed honey (such as ultra-high temperature sterilized products). Future exploration: