Semi-nested, multiplex polymerase chain reaction for detection of human malaria parasites and evidence of Plasmodium vivax infection in equatorial guinea
Semi-nested, multiplex polymerase chain reaction for detection of human malaria parasites and evidence of Plasmodium vivax infection in equatorial guinea
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DOI：
10.4269/ajtmh.1999.60.183
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发表时间：
1999-02-01
影响因子：
3.3
通讯作者：
Alvar, J
中科院分区：
文献类型：
--
作者：
Rubio, JM;Benito, A;Alvar, J
一种半巢式，多重聚合酶链反应（PCR）的基础上扩增的18S小亚基核糖体RNA（ssrRNA）基因的序列进行了测试，在赤道几内亚（疟疾在中西部非洲的高流行焦点）的现场试验。该方法使用了一种初级PCR扩增反应，其中使用了一种通用反向引物和两种对疟原虫属和哺乳动物具有特异性的正向引物（包括疟原虫特异性引物作为阳性对照，以区分未感染病例和PCR抑制）。第二次扩增使用相同的疟原虫属特异性正向引物和针对每个人疟原虫物种的四种特异性反向引物进行。在2%琼脂糖凝胶上电泳后，通过片段大小区分PCR扩增产物。对赤道几内亚比奥科岛三个地区的四个村庄和马拉博时间医院的两个疑似间日疟原虫卵圆型感染病例进行了显微镜和PCR检测。PCR方法显示出比显微镜检查更高的灵敏度和特异性，并证实了赤道几内亚存在一个通过显微镜检查怀疑的间日疟原虫感染病灶。它还提供了几种混合感染的证据，主要是恶性疟原虫和三日疟原虫，这是赤道几内亚两种主要的疟疾病原。
A semi-nested, multiplex polymerase chain reaction (PCR) based on the amplification of the sequences of the 18S small subunit ribosomal RNA (ssrRNA) gene was tested in a field trial in Equatorial Guinea (a hyperendemic focus of malaria in west central Africa). The method uses a primary PCR amplification reaction with a universal reverse primer and two forward primers specific for the genus Plasmodium and to mammals (the mammalian-specific primer was included as a positive control to distinguish uninfected cases from inhibition of the PCR). The second amplification is carried out with the same Plasmodium genus-specific forward primer and four specific reverse primers for each human Plasmodium species. The PCR amplified products are differentiated by fragment size after electrophoresis on a 2% agarose gel. Four villages from three regions of the island of Bioko (Equatorial Guinea) and two suspected Plasmodium vivax-P, ovale infections from time hospital of Malabo were tested by microscopy and PCR. The PCR method showed greater sensitivity and specificity than microscopic examination and confirmed the existence of a focus of P. vivax infections in Equatorial Guinea suspected by microscopic examination. It also provided evidence of several mixed infections, mainly P. falciparum and P. malariae, the two predominant species causing malaria in Equatorial Guinea.