建立一种用于检测孕妇、巨细胞病毒、单纯疱疹病毒 1 和 2 以及感染的实时多重聚合酶链反应方法。

Standardization of an in-house multiplex real-time polymerase chain reaction for the simultaneous detection of , cytomegalovirus, herpes simplex Virus 1 and 2, and infection among pregnant women.

机构信息

Sri Sakthi Amma Institute of Biomedical Research, Sri Narayani Hospital and Research Centre, Sripuram, Vellore, Tamil Nadu, India.

Environmental Molecular Microbiology Research Laboratory, Department of Biotechnology, Thiruvalluvar University, Serkadu, Vellore, Tamil Nadu, India.

出版信息

Indian J Public Health. 2021 Oct-Dec;65(4):369-374. doi: 10.4103/ijph.IJPH_1271_20.

DOI:10.4103/ijph.IJPH_1271_20

PMID:34975080

Abstract

BACKGROUND

An in-house multiplex real-time polymerase chain reaction (PCR) was developed in two cocktails for the identification of six Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and Treponema pallidum (syphilis) (TORCH-S) agents, which causes congenital infection among pregnant women.

OBJECTIVE

Standardization and validation of an in-house multiplex real-time PCR assay for the detection of TORCH-S infection.

METHODS

This study was conducted from February 2017 to February 2019. Primers specific for T. gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and T. pallidum were designed using Primer3 software (https://bioinfo.ut.ee/primer3-0.4.0/). The primer sequences obtained were subjected to BLAST analysis using BLAST database. Synthetic DNA was obtained to use as positive control templates for all the six TORCH-S agents. The lower limit of the detection was performed using plasmid construct for each virus serially diluted from 10 to 10.

RESULTS

An in-house multiplex real-time PCR was standardized and validated in two cocktails for TORCH-S agents, cocktail-1 (HSV1, rubella, and T. gondii), and cocktail-2 (HSV2, CMV, and T. pallidum). The lower limit of the detection for HSV1, rubella, and Toxoplasma were 60.7 copies/10 μl input, 76.4 copies/10 μl input, and 34.4 copies/10 μl input and for HSV2, CMV, and T. pallidum were 80.8 copies/10 μl input, 166 copies/10 μl input, and 43.7 copies/10 μl input, respectively.

CONCLUSION

TORCH-S infection is one of the significant reasons for irregular pregnant outcomes. It is absolutely important to screen TORCH-S infection for women who had the histories of abnormal pregnancies to prevent birth defects and perinatal complications. This multiplex real-time PCR assay provides a rapid, sensitive, and specific technique to detect these six TORCH-S agents.

摘要

背景

为了鉴定导致孕妇先天性感染的六种病原体（弓形虫、风疹病毒、巨细胞病毒、单纯疱疹病毒 1 和 2 型、梅毒螺旋体），我们开发了两种试剂盒的内部多重实时聚合酶链反应（PCR）。

目的

对用于检测 TORCH-S 感染的内部多重实时 PCR 检测进行标准化和验证。

方法

本研究于 2017 年 2 月至 2019 年 2 月进行。使用 Primer3 软件（https://bioinfo.ut.ee/primer3-0.4.0/）设计针对弓形虫、风疹病毒、巨细胞病毒、单纯疱疹病毒 1 和 2 型和梅毒螺旋体的特异性引物。获得的引物序列使用 BLAST 数据库进行 BLAST 分析。合成 DNA 用作所有六种 TORCH-S 病原体的阳性对照模板。使用质粒构建物从 10 到 10 对每个病毒进行连续稀释，以进行检测下限。

结果

我们标准化和验证了用于 TORCH-S 病原体的两种试剂盒的内部多重实时 PCR，试剂盒 1（HSV1、风疹和弓形虫）和试剂盒 2（HSV2、CMV 和梅毒螺旋体）。HSV1、风疹和弓形虫的检测下限分别为 60.7 拷贝/10 μl 输入、76.4 拷贝/10 μl 输入和 34.4 拷贝/10 μl 输入，HSV2、CMV 和梅毒螺旋体的检测下限分别为 80.8 拷贝/10 μl 输入、166 拷贝/10 μl 输入和 43.7 拷贝/10 μl 输入。