Khairnar Krishna, Parija Subhash C
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India.
BMC Microbiol. 2007 May 24;7:47. doi: 10.1186/1471-2180-7-47.
E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples.
The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR.
The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens.
溶组织内阿米巴是一种致病性阿米巴，通过光学显微镜观察，其包囊和滋养体阶段与非致病性的莫斯科维内阿米巴和迪斯帕内阿米巴难以区分。我们开发了一种巢式多重PCR，靶向类16S rRNA基因，用于在粪便样本中同时鉴别溶组织内阿米巴、莫斯科维内阿米巴和迪斯帕内阿米巴这三种形态相似的类型。
溶组织内阿米巴、莫斯科维内阿米巴和迪斯帕内阿米巴的种特异性产物大小分别为439 bp、553 bp和174 bp，这三种内阿米巴物种的产物大小明显不同。巢式多重PCR检测粪便样本中溶组织内阿米巴、莫斯科维内阿米巴和迪斯帕内阿米巴DNA的灵敏度为94%，特异性为100%。在通过显微镜检查和/或培养粪便检测溶组织内阿米巴/迪斯帕内阿米巴/莫斯科维内阿米巴呈阳性的202份粪便标本中，共有190份（灵敏度94%）对溶组织内阿米巴、莫斯科维内阿米巴和迪斯帕内阿米巴的PCR检测呈阳性。通过显微镜检查和培养，35份溶组织内阿米巴/迪斯帕内阿米巴/莫斯科维内阿米巴呈阴性的阴性对照粪便样本，经巢式多重PCR检测也呈阴性（特异性100%）。研究结果表明，通过显微镜检查和/或培养粪便检测溶组织内阿米巴/迪斯帕内阿米巴/莫斯科维内阿米巴呈阳性的患者粪便样本中，只有34.6%实际上为致病性溶组织内阿米巴阳性，其余大多数粪便样本通过巢式多重PCR检测表明为非致病性迪斯帕内阿米巴或莫斯科维内阿米巴阳性。
本研究报告了一种用于粪便标本中溶组织内阿米巴、迪斯帕内阿米巴和莫斯科维内阿米巴DNA种特异性检测和鉴别的新型巢式多重PCR策略。该检测具有高度特异性、灵敏性且快速，在收到粪便标本后12小时内即可得出结果。
