Gürses Gülcan, Yentür Doni Nebiye, Yıldız Zeyrek Fadile, Yiğin Akın
Harran University Vocational School of Health Services, Medical Laboratory Program Şanlıurfa, Turkey.
Harran University Faculty of Medicine, Department of Medical Microbiology, Şanlıurfa, Turkey.
Mikrobiyol Bul. 2022 Apr;56(2):326-338. doi: 10.5578/mb.20229811.
Cutaneous leishmaniasis (CL) is an important public health problem, most frequently seen in Şanlıurfa in Turkey. It is important to determine the species in regions where infection occurs with different Leishmania species, as in our province. In this study, it was aimed to genotype 136 samples with suspected Leishmania from Şanlıurfa using the Sybr Green-based ITS-1 real time polymerase chain reaction (Rt-PCR) method and then to compare them with ITS-1 PCR RFLP and direct microscopy methods. Wound fluid samples from patient lesions suspected of leishmaniasis were mounted on a slide, fixed, and stained with Giemsa dye. The preparations were examined under the microscope and evaluated for the presence of amastigote. After the extraction of DNA from Giemsa stained preparations by using the QIAmp DNA Mini Kit (Qiagen, Germany), the samples were studied with the Sybr Green based ITS-1 Rt-PCR method using LITSR and L5.8S primers. As a result of the PCR study, melting curve analysis was determined and the melting curves were compared with the reference strains. Then, PCR was performed in 136 samples for ITS1 region amplification using primers LITSR and L5.8S. PCR products were digested with Hae III restriction enzyme and RFLP process was performed. The products were run on metaphor agarose gel than the gels were stained with ethidium bromide for 15 min and visualized in a UV transilluminator In our study, the results of Sybr Green-based ITS-1 Rt-PCR, ITS-1 PCR-RFLP and direct microscopy methods were compared. The highest positivity rate was determined as 97% (136/132) in ITS-1 Rt-PCR method. With ITS-1 PCR-RFLP method 95.5% (136/130) positivity and with direct microscopy 94.1% (136/128) positivity were obtained, respectively. Of 132 samples, which were studied with the Sybr Green-based ITS-1 Rt-PCR method and found as positive, 121 were genotyped as L.tropica and 11 were genotyped as L.major by melting curve analysis. It was determined that, of 130 samples studied with ITS-1 PCR RFLP method 119 (91.5%) were detected as L.tropica and 11 (8.5%) were detected as L.major. The ITS-1 Rt-PCR method we used in our study was the method that detected the most positivity rate. With this method, Leishmania specimens were typed as L.tropica and L.major. It is thought that this method may be useful for the detection of the presence of Leishmania parasite and in the rapid identification of Leishmania species, as it does not require extra processes such as cutting and staining after PCR and results in a short time, but new studies are needed to observe its effectiveness in detecting other species other than L.tropica and L.major.
皮肤利什曼病（CL）是一个重要的公共卫生问题，在土耳其的尚勒乌尔法最为常见。在我们省份这样存在不同利什曼原虫物种感染的地区，确定物种很重要。本研究旨在使用基于SYBR Green的ITS-1实时聚合酶链反应（Rt-PCR）方法对来自尚勒乌尔法的136份疑似利什曼原虫样本进行基因分型，然后将其与ITS-1 PCR RFLP和直接显微镜检查方法进行比较。将疑似利什曼病患者病变的伤口液样本置于载玻片上，固定并用吉姆萨染料染色。在显微镜下检查制剂并评估无鞭毛体的存在。使用QIAmp DNA Mini试剂盒（德国Qiagen公司）从吉姆萨染色的制剂中提取DNA后，使用LITSR和L5.8S引物通过基于SYBR Green的ITS-1 Rt-PCR方法对样本进行研究。PCR研究的结果是确定熔解曲线分析，并将熔解曲线与参考菌株进行比较。然后，使用引物LITSR和L5.8S对136个样本进行ITS1区域扩增的PCR。PCR产物用Hae III限制性酶消化并进行RFLP过程。产物在琼脂糖凝胶上运行，然后凝胶用溴化乙锭染色15分钟，并在紫外透射仪中观察。在我们的研究中，比较了基于SYBR Green的ITS-1 Rt-PCR、ITS-1 PCR-RFLP和直接显微镜检查方法的结果。ITS-1 Rt-PCR方法的最高阳性率确定为97%（136/132）。ITS-1 PCR-RFLP方法的阳性率为95.5%（136/130），直接显微镜检查的阳性率为94.1%（136/128）。在用基于SYBR Green的ITS-1 Rt-PCR方法研究并发现为阳性的132个样本中，通过熔解曲线分析，121个被基因分型为热带利什曼原虫，11个被基因分型为硕大利什曼原虫。确定在使用ITS-1 PCR RFLP方法研究的130个样本中，119个（91.5%）被检测为热带利什曼原虫，11个（8.5%）被检测为硕大利什曼原虫。我们在研究中使用的ITS-1 Rt-PCR方法是检测阳性率最高的方法。用这种方法，利什曼原虫标本被分型为热带利什曼原虫和硕大利什曼原虫。人们认为这种方法可能有助于检测利什曼原虫寄生虫的存在并快速鉴定利什曼原虫物种，因为它不需要PCR后切割和染色等额外过程，且结果时间短，但需要新的研究来观察其在检测除热带利什曼原虫和硕大利什曼原虫以外的其他物种方面的有效性。
