Al-Quds Nutrition and Health Research Institute, Faculty of Medicine, Al-Quds University, Abu-Deis, West Bank, Palestine ; Institute of Microbiology and Hygiene, Charité University Medicine Berlin, Berlin, Germany.
PLoS Negl Trop Dis. 2013 Sep 26;7(9):e2464. doi: 10.1371/journal.pntd.0002464. eCollection 2013.
BACKGROUND/OBJECTIVES: Palestinian strains of L.tropica characterized by multilocus enzyme electrophoresis (MLEE) fall into two zymodemes, either MON-137 or MON-307.
METHODOLOGY/PRINCIPLE FINDINGS: Assays employing PCR and subsequent RFLP were applied to sequences found in the Hexokinase (HK) gene, an enzyme that is not used in MLEE, and the Phosphoglucomutase (PGM) gene, an enzyme that is used for MLEE, to see if they would facilitate consigning local strains of L.tropica to either zymodeme MON-137 or zymodeme MON-307. Following amplification and subsequent double digestion with the restriction endonucleases MboI and HaeIII, variation in the restriction patterns of the sequence from the HK gene distinguished strains of L.tropica, L.major and L.infantum and also exposed two genotypes (G) among the strains of L.tropica: HK-LtG1, associated with strains of L.tropica of the zymodemes MON-137 and MON-265, and HK-LtG2, associated with strains of L.tropica of the zymodemes MON-307, MON-288, MON-275 and MON-54. Following amplification and subsequent digestion by the restriction endonuclease MboI, variation in the sequence from the PGM gene also exposed two genotypes among the strains of L.tropica: PGM-G1, associated only with strains of L.tropica of the zymodeme MON-137; and PGM-G2, associated with strains of L.tropica of the zymodemes MON-265, MON-307, MON-288, MON-275 and MON-54, and, also, with six strains of L.major, five of L.infantum and one of L.donovani. The use of the HK and PGM gene sequences enabled distinction the L.tropica strains of the zymodeme MON-137 from those of the zymodeme MON-265. This genotyping system 'correctly' identified reference strains of L.tropica of known zymodemal affiliation and also from clinical samples, with a level of sensitivity down to <1 fg in the case of the former and to 1 pg of DNA in the case of the latter.
CONCLUSIONS/SIGNIFICANCE: Both assays proved useful for identifying leishmanial parasites in clinical samples without resource to culture and MLEE.
背景/目的：经多位点酶电泳（MLEE）鉴定的巴勒斯坦利什曼原虫株分为两种同工酶型，即 MON-137 或 MON-307。
方法/原理发现：应用聚合酶链反应（PCR）和随后的限制性片段长度多态性（RFLP）分析了己糖激酶（HK）基因和磷酸葡萄糖变位酶（PGM）基因的序列，这两种酶均不用于 MLEE，但用于同工酶型鉴定。这两种酶的序列是否有助于将当地的利什曼原虫株归属于同工酶型 MON-137 或同工酶型 MON-307。在扩增后，用限制性内切酶 MboI 和 HaeIII 进行双重消化，HK 基因序列的限制图谱变异可区分利什曼原虫、利什曼原虫和婴儿利什曼原虫的株系，并在利什曼原虫株系中暴露了两种基因型（G）：HK-LtG1，与同工酶型 MON-137 和 MON-265 的利什曼原虫株系相关；HK-LtG2，与同工酶型 MON-307、MON-288、MON-275 和 MON-54 的利什曼原虫株系相关。在扩增后，用限制性内切酶 MboI 消化，PGM 基因序列的变异也暴露了利什曼原虫株系中的两种基因型：PGM-G1，仅与同工酶型 MON-137 的利什曼原虫株系相关；PGM-G2，与同工酶型 MON-265、MON-307、MON-288、MON-275 和 MON-54 的利什曼原虫株系相关，也与 6 株利什曼原虫、5 株婴儿利什曼原虫和 1 株婴儿利什曼原虫相关。HK 和 PGM 基因序列的应用使同工酶型 MON-137 的利什曼原虫株系与同工酶型 MON-265 的利什曼原虫株系得以区分。这种基因分型系统“正确”地鉴定了已知同工酶归属的利什曼原虫参考株系，以及临床样本中的鉴定，前者的灵敏度低至<1 fg，后者的灵敏度低至 1 pg DNA。
结论/意义：这两种检测方法都有助于在无需培养和 MLEE 的情况下在临床样本中识别利什曼原虫寄生虫。
