PCR法检测粪便样本溶组织内阿米巴的初步研究

庞华胜; 匡紫微; 杨练; 孟妍明; 张春莹; 马莹

PCR法检测粪便样本溶组织内阿米巴的初步研究

Preliminary Study on PCR Method for Laboratory Diagnosis with Fecal Specimens of Entamoeba histolytica Infection

摘要

摘要:
目的建立直接检测粪便样本中溶组织内阿米巴的PCR方法。
方法根据溶组织内阿米巴标准株基因组中小亚基核糖体RNA（small subunit ribosome RNA，SSU rRNA）序列合成4对特异性引物（2对自主设计引物和2对参考引物），建立PCR检测方法，用该方法对221例临床腹泻患者粪便标本进行检测，同时采用病原学碘染涂片镜检法和酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)检测抗原，对3种方法的阳性率进行比较，并采用Kappa检验进行一致性分析，分析3种检测方法结果的准确性。
结果 4对引物均扩增出溶组织内阿米巴特异的目的片段，建立了粪便样本溶组织内阿米巴检测的PCR法。选择其中2对引物（1对自主设计引物和1对参考引物）对221例粪便样本进行PCR扩增，同时用碘染涂片镜检法查病原体，用ELISA法进行抗原检测，3种方法溶组织内阿米巴阳性检出率分别为2.26%、0.90%和9.50%，差异有统计学意义（χ²=23.34, P<0.01）。PCR法与碘染涂片镜检法比较，Kappa值为0.216，一致性微弱；PCR法与ELISA法比较，一致性差，Kappa值为–0.134。PCR法的结果与临床诊断的一致性最好。
结论本研究通过自行设计引物建立的粪便样本溶组织内阿米巴PCR检测法在准确性上优于镜检法和ELISA抗原检测法，为该方法用于临床诊断提供了实验基础。

Abstract:
Objective To develop a PCR method for Entamoeba histolytica（E.histolytica) detection in fecal specimens, and to compare the performance of PCR to that of microscopy and ELISA.
Methods Two pairs of self-designed primers and 2 pairs of primers from references based on small subunit ribosome RNA (SSU rRNA) fragment of E. histolytica standard strain were synthetized. DNA from E. histolytica reference strain were amilified by the conventional PCR using the 4 pairs of primers. 221 stool samples from diarrhea patients were collected and detected for E. histolytica by three methods: Entamoeba trophozoites and cysts detection by microscopy, E. histolytica-specific antigen detection using enzyme-linked immunosorbent assay (ELISA) kit (E. HISTOLYTICA II), amplification of SSU rRNA fragment of E. histolytica by PCR method. Positive rate of three methods were compared by chi-square test, and Kappa test was applied to determine the concordance among the three methods.
Results Specific fragments of E. histolytica were amplified by the PCR method we developed in this study. Positive rates of PCR, microscopy and ELISA were 2.26%, 0.90% and 9.50%, respectively. The positive rates of the three methods were significantly different (χ²=23.34, P<0.01). The Kappa value of PCR and microscopy was 0.216, and that of PCR and ELISA method was –0.134, both of which showed a weak consistency. PCR results showed best consistency with clinical diagnosis.
Conclusion The PCR method we established in this study has a better performance in accuracy than microscopy and ELISA have in laboratory diagnosis of E. histolytica infection.

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