Bagyalakshmi R, Therese K Lily, Madhavan H N
Larsen and Toubro Microbiology Research Centre, Vision Research Foundation, No. 18, College road, Sankara Nethralaya, Chennai-600006, India.
J Microbiol Methods. 2007 Jan;68(1):1-10. doi: 10.1016/j.mimet.2006.05.021. Epub 2006 Sep 7.
To analyse the genetic similarity among ocular isolates of Aspergillus flavus by Polymerase chain reaction based Restriction Fragment Length Polymorphism (PCR-RFLP) and DNA sequencing.
Seven ocular isolates of A. flavus from 5 patients (3 from paient 1, and four isolates from patients no. 2, 3, 4, and 5 respectively) consisting of 2 Aqueous Humor (AH), 2 Vitreous fluid (VF), 1 eviscerated material, 1 corneal button were included in the study. The three specimens from 1 were one each of AH, VF and corneal button. The fungal isolates were amplified using primers targeting ITS region and the amplicons were subjected to PCR-RFLP using Hae-III enzyme and DNA sequencing to analyse the genetic similarity.
A. flavus isolates yielded a specific product of 595 bp after amplification. All the seven A. flavus isolates showed similar pattern of digestion with Hae-III . However, DNA sequencing of ITS amplicons revealed 97.7% genetic similarity and 2.3% dissimilarity with nucleotide polymorphisms -- single, double and multiple pertaining to inversion, substitution, insertion and deletion in comparison with that of standard strain of A. flavus ATCC 16883 [Accession Number ]. A. flavus isolated from AH, VF and corneal button from the same patient showed similar nucleotide polymorphisms as against other isolates which exhibited distinct polymorphisms. This pattern of nucleotide polymorphisms in A. flavus isolates is novel and first time reported in literature to the best of our knowledge.
DNA sequencing proves to be a useful molecular tool in screening for nucleotide polymorphisms among fungal isolates.
通过基于聚合酶链反应的限制性片段长度多态性（PCR-RFLP）和DNA测序分析黄曲霉眼部分离株之间的遗传相似性。
本研究纳入了来自5例患者的7株黄曲霉眼部分离株（患者1的3株，患者2、3、4和5各1株），包括2份房水（AH）、2份玻璃体（VF）、1份眼球摘除物、1个角膜纽扣组织。患者1的3个标本分别为1份房水、1份玻璃体和1个角膜纽扣组织。使用靶向ITS区域的引物对真菌分离株进行扩增，扩增产物使用Hae-III酶进行PCR-RFLP分析并进行DNA测序，以分析遗传相似性。
黄曲霉分离株扩增后产生了一条595 bp的特异性产物。所有7株黄曲霉分离株用Hae-III酶消化后显示出相似的模式。然而，ITS扩增产物的DNA测序显示，与黄曲霉标准菌株ATCC 16883[登录号]相比，遗传相似性为97.7%，核苷酸多态性（涉及倒位、取代、插入和缺失的单、双和多个多态性）导致的差异为2.3%。从同一患者的房水、玻璃体和角膜纽扣组织中分离出的黄曲霉显示出与其他分离株不同的相似核苷酸多态性，其他分离株表现出明显的多态性。据我们所知，黄曲霉分离株中的这种核苷酸多态性模式是新颖的，首次在文献中报道。
DNA测序被证明是筛选真菌分离株中核苷酸多态性的一种有用的分子工具。
