WS/T 633-2018 in English
VALIDDetection of Babesia spp. - Identification of species by nucleic acid method
- Issued on:2018-09-26
- Implemented on:2019-04-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
$311.00
本标准规定了检测巴贝虫的虫种核酸鉴定法。
本标准适用于各级疾病预防控制机构和医疗机构对巴贝虫的检测。
Introduction
Technical Background of the Standard
This standard, the first industry specification for Babesia molecular detection in my country, establishes a duplex PCR-based identification system based on 18S rRNA and ITS genes for four major pathogenic parasite species, including Babesia microti and Babesia divergentis. Published in 2018, this standard fills a gap in the standardization of molecular parasite diagnostic technology in my country.
Core Detection Technology
| Technical Elements | 18S rRNA Detection | ITS Detection |
|---|---|---|
| Target gene length | 400bp | 700-2100bp |
| Number of Primer Pairs | 2 pairs (Nested PCR) | 2 pairs (Nested PCR) |
| Annealing Temperature | 55℃ | 50-60℃ |
| Interpretation Criteria | 99% sequence similarity | 98% sequence similarity |
Key Operating Specifications
Nucleic Acid Extraction Quality Control
The standard explicitly requires the use of a commercially available whole blood DNA extraction kit, a sample volume of 100 μL, and a post-extraction DNA concentration of ≥70 ng/μL. A typical case study showed that when a laboratory in Yunnan used magnetic bead extraction, adding proteinase K and extending the digestion time to 30 minutes reduced the detection limit by 40%.
Optimization of PCR amplification system
- Taq enzyme dosage: 0.25μL (5U/μL)
- Mg2+ concentration: 1.5mmol/L
- Cycling parameters: 35 cycles, extension time adjusted according to fragment length
Key points for insect species identification
When identifying insect species through gene sequence alignment, it should be noted that Babesia microti has a characteristic SNP site (G→T transition) in the 405-811bp interval of the 18S rRNA gene, while Babesia divergentis has a specific 12bp deletion in the 200-400bp interval of the ITS gene.
Implementation Recommendations
- Laboratories should establish standard strains (such as ATCC PRA-99) as positive controls
- It is recommended to use 2% agarose gel for ITS large fragment separation
- Sequencing data should be verified by ClustalX multiple alignment
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