PCR和LAMP检测阴道毛滴虫的研究

doi:10.12140/j.issn.1000-7423.2021.02.023

中国寄生虫学与寄生虫病杂志 ›› 2021, Vol. 39 ›› Issue (2): 260-264.doi: 10.12140/j.issn.1000-7423.2021.02.023

PCR和LAMP检测阴道毛滴虫的研究

赵丽丽¹(), 缪徐¹, 谢换飞¹, 刘碧涵¹, 袁琳¹, 林琳¹, 黄霜¹, 姜旭淦¹, 陈盛霞¹^,^*(), 沈玉娟², 曹建平²

1 江苏大学医学院检验医学系,镇江 212013
2 中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心）,国家卫生健康委员会寄生虫病原与媒介生物学重点实验室（中国疾病预防控制中心寄生虫病预防控制所）,上海 200025

收稿日期:2020-08-20 修回日期:2020-11-15 出版日期:2021-04-30 发布日期:2021-04-30
通讯作者: 陈盛霞
作者简介:赵丽丽（1998-）,女,学士,技师,从事临床检验研究。E-mail： 332772493@qq.com
基金资助:
国家寄生虫资源库(TDRC-2019-194-30);国家科技重大专项(2018ZX10713001-004)

Detection of Trichomonas vaginalis by PCR and LAMP

ZHAO Li-li¹(), MIAO Xu¹, XIE Huan-fei¹, LIU Bi-han¹, YUAN Lin¹, LIN Lin¹, HUANG Shuang¹, JIANG Xu-gan¹, CHEN Sheng-xia¹^,^*(), SHEN Yu-juan², CAO Jian-ping²

1 Department of Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang 212013, China
2 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research); NHC Key Laboratory of Parasite and Vector Biology (National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention), Shanghai 200025, China

Received:2020-08-20 Revised:2020-11-15 Online:2021-04-30 Published:2021-04-30
Contact: CHEN Sheng-xia
Supported by:
National Parasitic Resource Center(TDRC-2019-194-30);National Science and Technology Major Project(2018ZX10713001-004)

摘要/Abstract

摘要：

合成PCR和LAMP引物,分别扩增体外纯培养的阴道毛滴虫虫体DNA,建立阴道毛滴虫PCR和LAMP检测方法。通过检测临床镜检阴道毛滴虫阳性和阴性的阴道分泌物样品各40份,比较两种方法在临床应用的可行性;通过扩增解脲支原体、蓝氏贾第鞭毛虫、金黄色葡萄球菌、白假丝酵母菌、大肠埃希菌等的DNA,评价2种方法的特异性;测定体外纯培养的阴道毛滴虫虫体DNA浓度,经倍比稀释后,评价2种方法的灵敏度。结果显示,阴道毛滴虫DNA扩增后,PCR方法可检测出263 bp特异条带;LAMP方法显示扩增体系颜色由红色变为黄色,琼脂糖凝胶电泳可见DNA梯状条带。可行性检测结果显示,PCR检测40份临床阴道毛滴虫阳性样品和40份阴性样品的符合率分别为100%和100%,LAMP方法的符合率分别为100%和97.5%。特异性检测结果显示,2种方法均无法扩增其他5种病原体的DNA。灵敏度检测结果显示,PCR和LAMP方法的检测下限分别为0.5006 ng/μl和0.05006 ng/μl。提示PCR和LAMP技术都可用于阴道毛滴虫的检测,两种方法的特异性都较高,LAMP方法的灵敏度优于PCR方法。

关键词: 阴道毛滴虫, PCR, LAMP

Abstract:

To establish PCR and LAMP method for detection of Trichomonas vaginalis, the primers were synthesized, and the DNA of cultured of T. vaginalis was amplified. The feasibility of the two methods in clinical application was compared by detecting 40 T. vaginalis-positive and 40 T. vaginalis-negative samples of vaginal discharge identified by clinical microscopy. The specificity of the two methods was analyzed by amplifying DNA of Mycoplasma urealytium, Giardia lamblia, Staphylococcus aureus, Candida albicans and Escherichia coli. The sensitivity of the two methods was analyzed by determining the concentration of cultured T. vaginalis DNA after serial dilution. The results showed that PCR amplification of T. vaginalis DNA resulted in a 263-bp specific band, and the LAMP method caused a change of amplification product color from red to yellow, presenting the DNA ladder on agarose gel electrophoresis. The feasibility results showed that the coincidence rate of the 40 positive and 40 negative clinical samples by PCR were 100% and 100%, while LAMP method resulted in 100% and 97.5%, respectively. The specificity results showed that the DNA of 5 other pathogens could not be amplified by the two methods. The sensitivity test showed that the lower detection limit of the PCR method was 0.5006 ng/μl and the LAMP method 0.05006 ng/μl. These results indicate that the both methods are highly specific and the LAMP is superior to PCR in sensitivity.

Key words: Trichomonas vaginalis, PCR, LAMP

中图分类号:

R382.211

赵丽丽, 缪徐, 谢换飞, 刘碧涵, 袁琳, 林琳, 黄霜, 姜旭淦, 陈盛霞, 沈玉娟, 曹建平. PCR和LAMP检测阴道毛滴虫的研究[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(2): 260-264.

ZHAO Li-li, MIAO Xu, XIE Huan-fei, LIU Bi-han, YUAN Lin, LIN Lin, HUANG Shuang, JIANG Xu-gan, CHEN Sheng-xia, SHEN Yu-juan, CAO Jian-ping. Detection of Trichomonas vaginalis by PCR and LAMP[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2021, 39(2): 260-264.

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PCR和LAMP检测阴道毛滴虫的研究

Detection of Trichomonas vaginalis by PCR and LAMP

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