研究简报

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

  • 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
展开
  • 1 江西省寄生虫病防治研究所,南昌 330096
    2 深圳市康百得生物科技有限公司,广东深圳 518057
    3 江西省职业病防治研究院,南昌 330006
    4 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
兰炜明(1981-),男,硕士,副研究员,从事寄生虫病诊断与防治。E-mail:wmlan0795@163.com
*曾小军(1964-),男,本科,从事寄生虫病防治研究。E-mail:zengxiaojunnc@163.com

收稿日期: 2023-01-14

  修回日期: 2023-05-10

  网络出版日期: 2023-09-06

基金资助

江西省重点实验室计划项目(20192BCD40006);江西省自然科学基金项目(20212BAB206074);江西省卫生健康委科技计划(20204865)

Study on early warning of high risk environment of Schistosoma japonicum infection by quantitative real-time PCR

  • LAN Weiming ,
  • XU Hui ,
  • XU Yin ,
  • QIU Tingting ,
  • XIE Shuying ,
  • DENG Fenglin ,
  • HU Shaoliang ,
  • LIU Huan ,
  • GUO Jiagang ,
  • ZENG Xiaojun
Expand
  • 1 Jiangxi Provincial Institute of Parasitic Diseases, Nanchang 330096, China
    2 Shenzhen Combined Biotech Co. Ltd., Shenzhen 518057, Guangdong, China
    3 Institute of Occupational Medicine of Jiangxi, Nanchang 330006, China
    4 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 date: 2023-01-14

  Revised date: 2023-05-10

  Online published: 2023-09-06

Supported by

Jiangxi Province Key Lab Project(20192BCD40006);Jiangxi Natural Science Foundation(20212BAB206074);Science and Technology Plan of Jiangxi Provincial Health Commission(20204865)

摘要

采用实时荧光定量PCR(qRT-PCR)法检测感染早期哨鼠内脏组织日本血吸虫DNA,探索最佳检测时间,以达到早期监测预警血吸虫感染高危环境的目的。取20只阳性钉螺置于25 ℃温水中1 h逸出血吸虫尾蚴。将昆明小鼠随机分为自然感染组(6只)、定量感染组(36只)和阴性对照组(9只)。自然感染组小鼠转移至含有血吸虫尾蚴水体的笼内模拟野外现场自然感染1 h,定量感染组小鼠经腹部贴片法感染尾蚴(40 ± 5)条/鼠,阴性对照组小鼠不作感染。自然感染组小鼠感染后第1、2、5天随机解剖2只,定量感染组小鼠感染后第1、2、3、5、7、14天随机解剖6只,阴性对照组小鼠同步随机剖检1只。取各组小鼠腹部皮肤和心、肺、肝组织,提取DNA后进行qRT-PCR检测血吸虫DNA,记录循环阈值(Ct值),判断各组小鼠血吸虫感染情况;定量感染组小鼠还需单独检测个体各时间段肺组织中血吸虫DNA的Ct值。qRT-PCR检测结果显示,自然感染组小鼠腹部皮肤组织中血吸虫DNA在感染后第1、2天检测结果为阳性,肺组织在感染后第1、2、5天检测结果均为阳性,肝组织和心脏组织在感染后第5天为阳性。定量感染组小鼠皮肤在感染后第1、2、3天检测结果为阳性,肺组织在感染后所有时间点均为阳性,肝组织在感染后第2~14天均为阳性,心脏组织在感染后第3天和第7天为阳性,血液样品在感染后第5天为阳性。定量感染组小鼠个体肺组织在感染后第2~5天检测结果为阳性。提示哨鼠感染早期对其肺组织进行qRT-PCR检测血吸虫DNA,具有早期预警血吸虫感染高危环境的价值。

本文引用格式

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 . DOI: 10.12140/j.issn.1000-7423.2023.04.018

Abstract

To rapidly monitor and warn high-risk environments with Schistosoma japonicum, detecting S. japonicum DNA in the visceral tissues of sentinel rats at the early stage of infection by quantitative real-time PCR (qRT-PCR). 20 infected Oncomelania hupensis were placed in 25 ℃ warm water to release cercariae. The rats (KM species) were divided into three groups randomly, i.e. Group A with natural cercarial infection, Group B with quantitative cercarial infection and Group C without cercarial infection as a negative control group. In Group A, 6 rats were transferred into the cage with the water with cercariae for 1 h, which imitate the natural infection field. 36 rats in Group B were infected with cercariae (40 ± 5 per mouse) via the abdominal skin route. 9 rats in Group C were not infected with cercariae. The rats in Group A were anatomized randomly on day 1, 2 and 5 after infection. The rats in Group B were anatomized randomly on day 1, 2, 3, 5, 7 and 14 after infection, respectively. The rats in Group C were randomly selected to anatomized synchronously with the Group A and B. The tissues of abdominal skin, heart, lung and liver of each rat were collected after the dissection. Then the genomic DNA of the total collected tissues was extracted and tested for schistosome DNA through qRT-PCR. The Ct values were recorded to determine the infection rates in each group, especially for Group B, in which each rat's DNA Ct value of the lung tissue needed to record at different time period. As the qRT-PCR results showed, schistosome DNA in Group A was detected in lung on day 1, 2 and 5 after infection, and in liver on day 5 after infection. While in group B, the schistosome DNA was detected in the lung tissue on day 1, 2, 3, 5, 7 and 14 after infection, and later in the liver tissue on day 2, 3, 5, 7 and 14. It showed that the schistosome DNA in lung in group B can be detected from the 2nd to the 5th day after infection. The results suggested that the detection of schistosome DNA in the lung tissue by qRT-PCR at the early stage of sentinel rat infection has the value for early monitoring and warning of the high-risk environment with schistosome infection.

参考文献

[1] Zhou XN, Bergquist R, Leonardo L, et al. Schistosomiasis japonica control and research needs[J]. Adv Parasitol, 2010, 72: 145-178.
[2] He P, Gordon CA, Williams GM, et al. Real-time PCR diagnosis of Schistosoma japonicum in low transmission areas of China[J]. Infect Dis Poverty, 2018, 7: 8.
[3] Li SZ, Xu J, Wang TP, et al. Upholding Chinese spirit on schistosomiasis control in the new era to accelerate the progress towards schistosomiasis elimination in China[J]. Chin J Schisto Control, 2019, 31(1): 1-13. (in Chinese)
  (李石柱, 许静, 汪天平, 等. 弘扬新时期血防精神推进血吸虫病消除进程[J]. 中国血吸虫病防治杂志, 2019, 31(1): 1-13.)
[4] Chen C, Guo QH, Fu ZQ, et al. Reviews and advances in diagnostic research on Schistosoma japonicum[J]. Acta Trop, 2021, 213: 105743.
[5] Wu F, Huang YX. Application of determination of infested water in schistosomiasis forecast and early warning[J]. Chin J Schisto Control, 2010, 22(5): 500-503. (in Chinese)
  (吴锋, 黄轶昕. 疫水测定在血吸虫病预测预警中的应用[J]. 中国血吸虫病防治杂志, 2010, 22(5): 500-503.)
[6] Chen L, Cao CL, Liu Y, et al. Emergency responses to schistosomiasis outbreak during the stage moving towards elimination in China[J]. Chin J Schisto Control, 2021, 33(6): 570-574. (in Chinese)
  (陈琳, 曹淳力, 刘阳, 等. 迈向消除阶段我国血吸虫病突发疫情应急响应[J]. 中国血吸虫病防治杂志, 2021, 33(6): 570-574.)
[7] Cao CL, Li SZ, Zhou XN. Impact of schistosomiasis transmission by catastrophic flood damage and emergency response in China[J]. Chin J Schisto Control, 2016, 28(6): 618-623. (in Chinese)
  (曹淳力, 李石柱, 周晓农. 特大洪涝灾害对我国血吸虫病传播的影响及应急处置[J]. 中国血吸虫病防治杂志, 2016, 28(6): 618-623.)
[8] Qu GL, Dai JR, Xing YT, et al. Surveillance and forecast system of schistosomiasis in Jiangsu Province Ⅵ detection technology of water infectivity based on enrichment of Schistosoma japonicum cercariae on water surface[J]. Chin J Schisto Control, 2014, 26(5): 510-513. (in Chinese)
  (曲国立, 戴建荣, 邢云天, 等. 江苏省血吸虫病监测预警系统的研究Ⅵ基于水面日本血吸虫尾蚴富集的水体感染性检测技术[J]. 中国血吸虫病防治杂志, 2014, 26(5): 510-513.)
[9] Grimes JET, Croll D, Harrison WE, et al. The roles of water, sanitation and hygiene in reducing schistosomiasis: a review[J]. Parasit Vectors, 2015, 8: 156.
[10] Yu Q. Techniques for identification and detection of Schistosoma cercaria[J]. Int J Med Parasit Dis, 2015, 42(3): 180-183, 188. (in Chinese)
  (余晴. 血吸虫尾蚴鉴定与检测技术研究现状[J]. 国际医学寄生虫病杂志, 2015, 42(3): 180-183, 188.)
[11] Chen GY, Yi DS, Chen SZ, et al. Improvement on the method of sticking Schistosoma cercariae[J]. Med J Chin PLA, 1965(1): 40-43. (in Chinese)
  (陈光裕, 易道善, 陈守正, 等. 粘取血吸虫尾蚴方法的改进[J]. 解放军医学杂志, 1965(1): 40-43.)
[12] Wang J, Yu CX, Yin XR, et al. Monitoring specific antibody responses against the hydrophilic domain of the 23 kDa membrane protein of Schistosoma japonicum for early detection of infection in sentinel mice[J]. Parasit Vectors, 2011, 4(1): 172.
[13] Zhang F, Hou M, Yu CX, et al. The use of nested PCR to monitor sentinel mice in aquatic areas infested with schistosomiasis japonica[J]. J Pathogen Biol, 2015, 10(4): 325-328. (in Chinese)
  (张凡, 侯敏, 余传信, 等. 巢式PCR法在日本血吸虫易感水域哨鼠监测中的应用[J]. 中国病原生物学杂志, 2015, 10(4): 325-328.)
[14] Deng WP, Wang SL, Wang LP, et al. Laboratory evaluation of a basic recombinase polymerase amplification (RPA) assay for early detection of Schistosoma japonicum[J]. Pathogens, 2022, 11(3): 319.
[15] Ullah H, Qadeer A, Giri BR. Detection of circulating cell-free DNA to diagnose Schistosoma japonicum infection[J]. Acta Trop, 2020, 211: 105604.
[16] Mu Y, Cai PF, Olveda RM, et al. Parasite-derived circulating microRNAs as biomarkers for the detection of human Schistosoma japonicum infection[J]. Parasitology, 2020, 147(8): 889-896.
[17] Ullah H, Arbab S, Khan MIU, et al. Circulating cell-free mitochondrial DNA fragment: a possible marker for early detection of Schistosoma japonicum[J]. Infect Genet Evol, 2021, 88: 104683.
[18] Peng GH, Wei WY, Qian K, et al. Study on rapid recognition technique of schistosome susceptibility water body Ⅰ animal skin making and effect assessment[J]. Chin J Schisto Control, 2018, 30(2): 200-201, 210. (in Chinese)
  (彭国华, 魏望远, 钱科, 等. 快速识别血吸虫易感性水体技术研究Ⅰ动物皮膜制作与检测效果初报[J]. 中国血吸虫病防治杂志, 2018, 30(2): 200-201, 210.)
[19] Mao SB. Biology of Schistosoma japonicum and its prevention and treatment[M]. Beijing: People's Medical Publishing House, 1990: 140-143. (in Chinese)
  (毛守白. 血吸虫生物学与血吸虫病的防治[M]. 北京: 人民卫生出版社, 1990: 140-143.)
[20] Hu HB, Xu GY. Observation on the infection of sentinel rats exposed to epidemic water at different times[J]. Parasit Infect Dis, 1999(3): 138. (in Chinese)
  (胡鸿宝, 徐国余. 哨鼠接触疫水不同时间感染情况观察[J]. 实用寄生虫病杂志, 1999(3): 138.)
文章导航

/

〈 〉