收稿日期: 2023-09-14
修回日期: 2024-01-09
网络出版日期: 2024-03-12
基金资助
武汉市卫生计生委医学科研项目(WG17B04);武汉市卫生计生委医学科研项目(WG18Q05);武汉市卫生健康委第七批中青年医学骨干人才培养工程
Establishment of loop-mediated isothermal amplification detection for Paragonimus westermani
Received date: 2023-09-14
Revised date: 2024-01-09
Online published: 2024-03-12
Supported by
Wuhan Municipal Health and Family Planning Commission Medical Research Project(WG17B04);Wuhan Municipal Health and Family Planning Commission Medical Research Project(WG18Q05);Seventh Batch of Young and Middle-aged Medical Backbone Talent Training Projects of Wuhan Municipal Health and Health Commission
2022年于卫氏并殖吸虫流行区湖北兴山县和保康县分别采集34只和27只溪蟹，捣碎后用NaOH消化法提取DNA。根据卫氏并殖吸虫5.8S核糖体RNA序列，使用Primer Explorer V5软件设计卫氏并殖吸虫环介导等温扩增（LAMP）特异引物，建立卫氏并殖吸虫LAMP检测方法，评价该方法的特异性、灵敏度、扩增效率及现场应用效果，并与沉淀镜检法比较。结果显示，建立的LAMP检出限可达1个卫氏并殖吸虫囊蚴，且与日本血吸虫、华支睾吸虫、肝片形吸虫、肝毛细线虫、钩虫等虫种无交叉反应。检测4份卫氏并殖吸虫囊蚴DNA样品，出现浊度时间（Tt值）和浊度速率峰值（Df）均值为29.0 min和0.237。LAMP检出兴山和保康县卫氏并殖吸虫阳性溪蟹分别为24只和0只，阳性率分别为70.6%（24/34）和0（0/27）；沉淀镜检法检出囊蚴阳性溪蟹分别为23只和0只，阳性率分别为67.6%（23/34）和0（0/27），两种方法结果差异无统计学意义（χ2 = 0.2，P > 0.05）。建立的卫氏并殖吸虫LAMP检测方法操作简便、特异性强、灵敏度高，可用于淡水蟹卫氏并殖吸虫流行区现场调查或筛查。
周水茂 , 贾西帅 , 杨燕 , 张娟 . 卫氏并殖吸虫环介导等温扩增检测方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2024 , 42(1) : 121 -124 . DOI: 10.12140/j.issn.1000-7423.2024.01.018
In 2022, a total of 34 and 27 brook crabs were collected from Xingshan and Baokang Counties, respectively, in Hubei Province, a region where Paragonimus westermani is endemic. The DNA was extracted by NaOH digestion method after crushing from crab. Based on the 5.8S ribosomal RNA sequence of P. westermani, Primer Explorer V5 software was used to design specific primers for loop-mediated isothermal amplification (LAMP) of P. westermani, and a LAMP detection method for P. westermani was established. The specificity, sensitivity, amplification efficiency, and field application effect of the method were evaluated, and compared with precipitation microscopy. The results indicated that the LAMP method displayed a detection limit of a single P. westermani metacercaria without any cross-reactivity with five non-target species, including Schistosoma japonicum, Clonorchis sinensis, Fasciola hepatica, Capillaria hepatica, and hookworm. The assay’s performance was quantified using the average turbidity time (Tt value) of 29.0 min and the peak turbidity rate (Df) of 0.237 min for four metacercariae samples. The LAMP method revealed 24 and 0 positive stream crabs infceted with P. westermani in Xingshan County and Baokang County, with positive rates of 70.6% (24/34) and 0 (0/27), respectively; the precipitation microscopy method revealed 23 and 0 positive stream crabsin Xingshan County and Baokang County, with positive rates of 67.6% (23/34) and 0 (0/27), respectively. There is no statistically significant difference in the results between the two detection methods (χ2 = 0.2, P > 0.05). Conclusively, the LAMP method developed in this study is a straightforward, specific, and highly sensitive diagnostic tool for the detection of P. westermani in freshwater crabs. Its proven efficacy suggests that it is well-suited for in-field surveys or as a screening mechanism in endemic regions.
| [1] | Zhu XP, Su C. Human parasitology[M]. 9th ed. Beijing: People’s Medical Publishing House, 2018: 96-101. (in Chinese) |
| (诸欣平, 苏川. 人体寄生虫学[M]. 9版. 北京: 人民卫生出版社, 2018: 96-101.) | |
| [2] | Utzinger J, Becker SL, Knopp S, et al. Neglected tropical diseases: diagnosis, clinical management, treatment and control[J]. Swiss Med Wkly, 2012, 142: w13727. |
| [3] | Rabone M, Wiethase J, Clark PF, et al. Endemicity of Paragonimus and paragonimiasis in Sub-Saharan Africa: a systematic review and mapping reveals stability of transmission in endemic foci for a multi-host parasite system[J]. PLoS Negl Trop Dis, 2021, 15(2): e0009120. |
| [4] | Nagayasu E, Yoshida A, Hombu A, et al. Paragonimiasis in Japan: a twelve-year retrospective case review (2001—2012)[J]. Intern Med, 2015, 54(2): 179-186. |
| [5] | Fürst T, Keiser J, Utzinger J. Global burden of human food-borne trematodiasis: a systematic review and meta-analysis[J]. Lancet Infect Dis, 2012, 12(3): 210-221. |
| [6] | Liu Q, Wei F, Liu WS, et al. Paragonimiasis: an important food-borne zoonosis in China[J]. Trends Parasitol, 2008, 24(7): 318-323. |
| [7] | Chen JX. Food-borne parasitic disease[M]. Beijing: People’s Medical Publishing House, 2009: 58-66. (in Chinese) |
| (陈家旭. 食源性寄生虫病[M]. 北京: 人民卫生出版社, 2009: 58-66.) | |
| [8] | Wang TY, Ye KL, Jiao X. Research progress on visual detection of nucleic acids techniques[J]. Biol Chem Eng, 2023, 9(1): 163-166, 183. (in Chinese) |
| (王天予, 叶凯霖, 焦雪. 核酸可视化检测技术研究进展[J]. 生物化工, 2023, 9(1): 163-166, 183.) | |
| [9] | Mahmoudi MR, Kazemi B, Mohammadiha A, et al. Detection of Cryptosporidium and Giardia (oo)cysts by IFA, PCR and LAMP in surface water from Rasht, Iran[J]. Trans R Soc Trop Med Hyg, 2013, 107(8): 511-517. |
| [10] | Ai L, Li C, Elsheikha HM, et al. Rapid identification and differentiation of Fasciola hepatica and Fasciola gigantica by a loop-mediated isothermal amplification (LAMP) assay[J]. Vet Parasitol, 2010, 174(3/4): 228-233. |
| [11] | Zhang J, Xia J, Zhang HX, et al. Surveillance on Paragonimus infection in Hubei Province from 2018 to 2020[J]. Chin J Parasitol Parasit Dis, 2021, 39(5): 600-605. (in Chinese) |
| (张娟, 夏菁, 张华勋, 等. 2018—2020年湖北省并殖吸虫感染监测结果分析[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(5): 600-605.) | |
| [12] | Gong ZH, Gong HK, Xu Y, et al. Retrospective analysis of paragonimiasis cases in Jiangxi Province from 2011 to 2020[J]. Chin J Parasitol Parasit Dis, 2022, 40(2): 247-251. (in Chinese) |
| (龚志红, 龚红卡, 徐芸, 等. 江西省2011—2020年并殖吸虫病病例回顾性分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(2): 247-251.) | |
| [13] | Li RZ, Chen L, Xu L, et al. Surveillance results of paragonimiasis in Sichuan Province from 2015 to 2020[J]. J Prev Med Inf, 2022, 38(11): 1435-1440. (in Chinese) |
| (李荣智, 陈琳, 徐亮, 等. 2015—2020年四川省并殖吸虫监测结果分析[J]. 预防医学情报杂志, 2022, 38(11): 1435-1440.) | |
| [14] | Lou HQ, Hu Y, Jin YJ, et al. Investigation on the hosts with natural Paragonimus infection and species identification in Jinhua prefecture of Zhejiang Province[J]. Chin J Parasitol Parasit Dis, 2011, 29(5): 348-352. (in Chinese) |
| (楼宏强, 胡野, 金耀建, 等. 浙江省金华地区并殖吸虫自然宿主调查及虫种鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2011, 29(5): 348-352.) | |
| [15] | General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China. Quarantine protocol for metacercaria of Paragonimus in crustaceans: SN/T 3504—2013[S]. Beijing: China Standard Publishing House, 2013: 4-6. (in Chinese) |
| (中华人民共和国国家质量监督检验检疫总局. 甲壳类水产品中并殖吸虫囊蚴检疫技术规范: SN/T 3504—2013[S]. 北京: 中国标准出版社, 2013: 4-6.) | |
| [16] | Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res, 2000, 28(12): E63. |
| [17] | Han ET, Watanabe R, Sattabongkot J, et al. Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis[J]. J Clin Microbiol, 2007, 45(8): 2521-2528. |
| [18] | Kuboki N, Inoue N, Sakurai T, et al. Loop-mediated isothermal amplification for detection of African trypanosomes[J]. J Clin Microbiol, 2003, 41(12): 5517-5524. |
| [19] | Mazidur Rahman SM, Song HB, Jin Y, et al. Application of a loop-mediated isothermal amplification (LAMP) assay targeting cox1 gene for the detection of Clonorchis sinensis in human fecal samples[J]. PLoS Negl Trop Dis, 2017, 11(10): e0005995. |
| [20] | Liu W, Jin JH, Chen XR. Application progress of loop-mediated isothermal amplification technique[J]. Curr Biotechnol, 2021, 11(2): 128-135. (in Chinese) |
| (刘旺, 靳晶豪, 陈孝仁. 环介导等温扩增技术的应用进展[J]. 生物技术进展, 2021, 11(2): 128-135.) |
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