多重巢式聚合酶链反应在疟疾混合感染检测与分型中的应用

摘要

Objective:To develop a multiplex nested PCR method for detecting of P.falciparum, P.vivax, P. ovale,P.malarie and mixed Plasmodium malariae.And to evaluate the application value of multiplex nested PCR in the diagnosis and typing of malaria.In order to supply an effective supplement of microscopic examination for the detection and epidemiology of malaria.Methods: 5 pairs of primers ( 1 pair of universal primers and 4 pairs of specific primers) of the 4 kinds of malarie were designed and synthesized based on the 18 SSU rRNA to develop the multiplex nested PCR.28 blood samles were detected by nested PCR and compare the results with microscopic examination.Results: Nested PCR results showed that 6 samples were mixed Plasmodium malariae infection,consistant with sequencing.Conclusion: We have successfully developed a multiplex nested PCR method for dececting and typing of P.Falciparum, P.Vivax, P.Ovale, P.Malarie and mixed Plasmodium malariae.It shows higher sensitivity and specificity than microscopic examination on detecting and typing of malaria.%目的：应用能够同时检测恶性疟（ P.falciparum）、间日疟（ P.vivax）、卵形疟（ P.ovale）、三日疟（ P.malarie）及其混合感染的多重巢式PCR方法，对疑似疟疾混合感染样本进行检测，评价其在疟原虫混合感染及分型中的应用价值，旨在为疟疾流行病学调查、疟疾混合感染的确诊提供一种快速高效、特异性强、灵敏度高的实验室诊断方法，为传统的镜检法提供有效的补充。方法：根据恶性疟原虫、间日疟原虫、卵形疟原虫和三日疟原虫的18 SSU rRNA基因序列设计5对引物（1对通用引物、4对特异性引物），以等比例混合的4种疟原虫核酸为模板，建立疟疾多重巢式PCR检测方法，并应用该法对28份疑似疟疾混合感染样本进行检测及测序验证，将其结果与镜检法比较。结果：28份疑似混合感染样本中混合感染样品数为6例，其中恶性疟和间日疟混合感染样品5例，恶性疟和卵形疟混合感染样品1例。结论：本研究建立的多重巢式PCR检测方法能够同时用于恶性疟、间日疟、卵形疟、三日疟及其混合感染样本的检测，在疟疾混合感染的确诊与分型上较镜检法有明显的优势和更高的应用价值。