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.

图/表 6

图1

图2

图3

图4

图5

图6

参考文献 18

[1]	Aquino M, Hinderfeld AS, Simoes-Barbosa A. Trichomonas vaginalis[J]. Trends Parasitol, 2020,36(7):646-647.
[2]	Edwards T, Burke P, Smalley H, et al. Trichomonas vaginalis: clinical relevance, pathogenicity and diagnosis[J]. Crit Rev Microbiol, 2016,42(3):406-417.
[3]	Noh CS, Kim SS, Park SY, et al. Comparison of two PCR assays for Trichomonas vaginalis[J]. Korean J Parasitol, 2019,57(1):27-31.
[4]	Lan L, Wang PP, Fang DD, et al. Study on polymerase chain reaction for detection of Trichomonas vaginalis[J]. J Changzhi Med Coll, 2015,29(6):409-412. (in Chinese)
[4]	( 兰龙, 王盼盼, 方丹丹, 等. 聚合酶链反应技术检测阴道毛滴虫的研究[J]. 长治医学院学报, 2015,29(6):409-412.)
[5]	Rumyantseva T, Golparian D, Nilsson CS, et al. Evaluation of the new AmpliSens multiplex real-time PCR assay for simultaneous detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis[J]. APMIS, 2015,123(10):879-886.
[6]	Leli C, Castronari R, Levorato L, et al. Molecular sensitivity threshold of wet mount and an immunochromatographic assay evaluated by quantitative real-time PCR for diagnosis of Trichomonas vaginalis infection in a low-risk population of childbearing women[J]. Infez Med, 2016,24(2):112-116.
[7]	Ziaei HH, Taghavi M, Fakhar M, et al. Direct diagnosis of Trichomonas vaginalis infection on archived pap smears using nested PCR[J]. Acta Cytol, 2015,59(1):104-108.
[8]	Reyes JC, Solon JA, Rivera WL. Development of a loop-mediated isothermal amplification assay for detection of Trichomonas vaginalis[J]. Diagn Microbiol Infect Dis, 2014,79(3):337-341.
[9]	Goo YK, Shin WS, Yang HW, et al. Loop-mediated isothermal amplification targeting actin DNA of Trichomonas vaginalis[J]. Korean J Parasitol, 2016,54(3):329-334.
[10]	Jiang HQ, Wei SS, Wu LM, et al. Comparison of PCR detection of different primers for Trichomonas vaginalis[J]. J Mod Med Health, 2015,31(22):3448-3450. (in Chinese)
[10]	( 江海强, 魏帅帅, 吴腊梅, 等. 阴道毛滴虫不同引物PCR检测结果比较[J]. 现代医药卫生, 2015,31(22):3448-3450.)
[11]	Adao DE, Rivera WL. Loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the sexually-transmitted parasite, Trichomonas vaginalis[J]. Ann Parasitol, 2016,62(1):25-31.
[12]	Fichorova R, Fraga J, Rappelli P, et al. Trichomonas vaginalis infection in symbiosis with Trichomonasvirus and Mycoplasma[J]. Res Microbiol, 2017,168(9/10):882-891.
[13]	Kuang GR, Yi FY, Yang YJ, et al. Investigation on symbiosis of Mycoplasma hominis in Trichomonas vaginalis in some areas of Guizhou province[J]. J Guiyang Med Coll, 2015,40(3):249-252. (in Chinese)
[13]	( 匡贵榕, 衣凤芸, 杨宇箭, 等. 贵州省部分地区阴道毛滴虫与人型支原体共生情况[J]. 贵阳医学院学报, 2015,40(3):249-252.)
[14]	Chen Y, Liu SL, Ma DX, et al. Analysis of 19 342 cases of Trichomonas vaginalis[J]. J Trop Med, 2019,19(9):1177-1179. (in Chinese)
[14]	( 陈杨, 刘素伶, 马丹霞, 等. 19 342例阴道毛滴虫检查结果分析[J]. 热带医学杂志, 2019,19(9):1177-1179.)
[15]	Nikpay S, Otaghi M, Azami M, et al. Trichomonas vaginalis infection among women attending laboratory centers in Ilam, Iran[J]. Infect Disord Dug Targets, 2020,20(1):98-101.
[16]	Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP): recent progress in research and development[J]. J Infect Chemother, 2013,19(3):404-411.
[17]	Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases[J]. J Infect Chemother, 2009,15(2):62-69.
[18]	Yuhua L, Shuai W, Haoran L, et al. Development of a convenient detection method for Trichomonas vaginalis based on loop-mediated isothermal amplification targeting adhesion protein 65[J]. BMC Infect Dis, 2020,20:319-326.

PCR和LAMP检测阴道毛滴虫的研究

Detection of Trichomonas vaginalis by PCR and LAMP

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 18

相关文章 15

编辑推荐

Metrics

本文评价

[1]	唐琦, 吕超, 王熙, 郭苏影, 许晓娟, 朱海, 李银龙, 林伟娜, 周新杰, 冯婷, 许静, 秦志强. 3种方法检测野鼠血吸虫感染的效果评价[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(2): 186-191.
[2]	王真瑜, 江莉, 余晴, 张耀光, 吴寰宇, 陈健, 朱民. 《疟原虫核酸检测多重PCR法》标准解读[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(4): 521-524.
[3]	周欣, 黄翠兰. 抗阴道毛滴虫靶点及药物研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(1): 105-110.
[4]	马志雅, 谢世臣, 贺渊惠, 高文伟, 刘卿, 朱兴全, 郑文斌. 山西省绵羊捻转血矛线虫的感染现状及遗传变异分析[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(6): 733-738.
[5]	原慧真, 李栋梁, 程书琪, 菅复春. 芽囊原虫体外培养特性的研究[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(5): 631-635.
[6]	徐银, 刘婷, 徐慧, 曾小军, 兰炜明, 龚志红, 戴坤教, 邱婷婷, 郝先, 谢曙英. PCR-CRISPR/Cas12a华支睾吸虫囊蚴检测方法的建立和应用[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 421-426.
[7]	蔡长煌, 张芝平, 卓鸣莺, 郭理平, 傅志辉, 刘亦若. 基于虫卵内转录间隔区2序列分析诊断麝猫后睾吸虫和华支睾吸虫感染[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 427-433.
[8]	黎玉琼, 于有利, 郜军荣, 刘芸芸, 李红兵, 牛晓昊. 宁夏银川地区奶牛毕氏肠微孢子虫的感染情况与基因型鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 476-479.
[9]	兰炜明, 徐慧, 徐银, 邱婷婷, 谢曙英, 邓凤林, 胡绍良, 刘欢, 郭家钢, 曾小军. 荧光定量PCR用于日本血吸虫感染高危环境早期预警的研究[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 502-505.
[10]	李美, 肖宁, 夏志贵. 基于无性期18S rDNA特异性引物检测5种疟原虫qPCR的建立和应用[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(1): 36-43.
[11]	刘继兵, 赵洪喜. 基于18S rRNA的胎毛滴虫感染PCR诊断方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(5): 682-685.
[12]	江莉, 张耀光, 刘红霞, 王真瑜, 朱民, 吴寰宇. 疟疾蚊媒监测多重PCR方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(2): 159-167.
[13]	罗澄扬, 郝利霞, 郭丽华, 王兵立, 纪思帆, 连安娜, 朱晓慧, 梅雪芳, 田晓薇, 王帅, 张振超. 阴道毛滴虫分泌蛋白对小鼠精子质量的影响[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(2): 236-241.
[14]	付梅花, 王旭, 韩帅, 喻文杰, 杨毅, 高明军, 刘剑峰, 官亚宜, 王莹, 李春阳, 施丹丹, 伍卫平. 四川省色达县野外棘球绦虫终末宿主粪便污染情况及特征[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(5): 687-695.
[15]	段玉娟, 李朋举, 桑雨慧, 吴璞成, 商雨佳, 郝利霞, 王帅, 李祥瑞, 张振超. 阴道毛滴虫感染对男性生殖系统的影响及其机制[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(4): 532-536.