GB/T 40249-2021 in English

VALID

Code of diagnosis for infection with Penaeus monodon-type baculovirus—PCR method

Issued on:2021-05-21
Implemented on:2021-12-01
File Format:PDF
Delivery:Via email within 1~3 business days

Price(USD)： $220.00
$214.00

Standard No:	GB/T 40249-2021
Document status:	VALID
Title in English:	Code of diagnosis for infection with Penaeus monodon-type baculovirus—PCR method
Title in Chinese:	斑节对虾杆状病毒病诊断规程 PCR检测法
Language:	English
File Format:	Electronic (PDF)
Delivery:	Via email within 1~3 business days
Issued on:	2021-05-21
Implemented on:	2021-12-01
ICS Classification:	65.020.30-Animal husbandry and breeding
Professional Classification:	GB-National Standard
Related Keywords:	monodon-type baculovirus pcr method introduction analysis penaeus monodon penaeus monodon seedlings pcr amplification system winter transportation avoid pcr product contamination
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《GB/T 40249-2021斑节对虾杆状病毒病诊断规程 PCR检测法》由TC156（全国水产标准化技术委员会）归口，TC156SC11（全国水产标准化技术委员会水产养殖病害防治分会）执行，主管部门为农业农村部。

Introduction

Analysis of the technical framework of the standard

Core elements	GB/T40249-2021 requirements	Comparison of similar standards
Detection objects	Tissue samples of Penaeus monodon at various growth stages	WSSV detection requires separate sampling
Primer specificity	261F/R targets 261bp conserved sequences	Most standards use a single pair of primers
Quality control system	Establish internal reference (848bp) + three-level control	Some standards lack internal references

Key technical points

1. Sample processing specifications

The standard stipulates that larvae should be sampled in their entirety, and adult shrimp should have their hepatopancreas and intestinal tissues separated. The samples need to be extracted with phenol-chloroform method for DNA extraction. The key steps include:

Proteinase K digestion: 50℃ treatment for 3 hours to ensure cell lysis
Organic phase separation: phenol/chloroform/isoamyl alcohol (25:24:1) to remove proteins
Ethanol precipitation: -20℃ precipitation for 2 hours to ensure DNA recovery

2. Optimization of PCR amplification system

The reaction system uses 1.5mM Mg²⁺ and 0.3μM primer concentration. The amplification program settings are:

Pre-denaturation at 94℃ for 5 minutes
35 cycles (94℃ 30s→60℃ 30s→72℃ 30s)
Final extension at 72℃ for 7 minutes

Implementation and application suggestions

Typical detection case

A farm used this standard to detect Penaeus monodon seedlings and found that:

Positive sample electrophoresis showed a clear band of 261bp
Sequencing results matched the reference sequence in Appendix C by >99%
Timely isolation and treatment to avoid the spread of the epidemic

Precautions

It is recommended to use safe dyes such as GelRed as an alternative to EB
Dry ice insulation is required for winter transportation
Avoid PCR product contamination causing false positives

Standard evolution analysis

Compared with the 2003 draft, this standard:

Add internal reference primers to control false negatives
Optimize annealing temperature to 60℃ to improve specificity
Clear sequencing verification requirements

More samples: DL/T 368-2010 GB 18306-2015 JC/T 2453-2018

Sample only — not a preview of GB/T 40249-2021

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