Akbarian Asiye, Shahhosseiny Mohammad Hassan, Vafaei Somayeh, Moslemi Elham, Ghahri Maryam
Department of Immunology, Tarbiat Modares University, Tehran, IR Iran.
Department of Microbiology, Shahre Qods, Branch Islamic Azad University, Tehran, IR Iran.
Jundishapur J Microbiol. 2015 Feb 20;8(2):e16260. doi: 10.5812/jjm.16260. eCollection 2015 Feb.
PCR is a molecular technique for herpes simplex virus (HSV) detection that can cause life-threatening infections such as encephalitis and keratitis. However, the main issues, false-negative results causing by PCR inhibitors, of this technique that reduce PCR efficiency. To overcome this problem, a competitive internal amplification control (IAC) was constructed for conventional PCR using the PCR-cloning technique.
The purpose of this study is the design of competitive IAC for PCR diagnosis of HSV, which in fact is the main cause of keratitis and viral encephalitis in developed countries.
Composite primers for PCR amplification of Leishmania major kDNA (kinetoplast DNA) were designed and optimized to use as IAC-HSV. IAC-HSV amplified in a non-stringent condition, ligated into pTZ57R plasmid vector, and transformed into Escherichia coli JM207 and then cloned. Resulting IAC was used for 105 CSF and 78 keratitis specimens.
PCR amplicons for HSV and IAC-HSV were 454-bp and 662-bp, respectively. Detection limit of IAC was determined as 1000 plasmids per PCR reaction. IAC sensitivity for HSV detection was determined as 1000 plasmids per PCR reaction. IAC sensitivity for HSV detection was 500 copies/mL of HSV DNA. Among all specimens, 7 inhibited specimens were detected.
Indeed, using other DNA as an IAC is expected to detect false-negative results and amplification of the DNA is the key tool to examine the accuracy of amplification and detection steps. This internal amplification control is applicable for early reliable diagnosis of HSV in different loads of virus in different specimens.
聚合酶链反应（PCR）是一种用于检测单纯疱疹病毒（HSV）的分子技术，该病毒可引发如脑炎和角膜炎等危及生命的感染。然而，这项技术存在主要问题，即PCR抑制剂会导致假阴性结果，从而降低PCR效率。为克服这一问题，利用PCR克隆技术构建了用于常规PCR的竞争性内参扩增对照（IAC）。
本研究旨在设计用于HSV PCR诊断的竞争性IAC，HSV实际上是发达国家角膜炎和病毒性脑炎的主要病因。
设计并优化用于利什曼原虫主要动质体DNA（kDNA）PCR扩增的复合引物，用作IAC-HSV。IAC-HSV在非严格条件下扩增，连接到pTZ57R质粒载体中，转化到大肠杆菌JM207中，然后进行克隆。所得IAC用于105份脑脊液标本和78份角膜炎标本。
HSV和IAC-HSV的PCR扩增产物分别为454 bp和662 bp。IAC的检测限确定为每个PCR反应1000个质粒。IAC对HSV检测的灵敏度确定为每个PCR反应1000个质粒。IAC对HSV检测的灵敏度为500拷贝/毫升HSV DNA。在所有标本中，检测到7份抑制标本。
实际上，使用其他DNA作为IAC有望检测出假阴性结果，而DNA的扩增是检验扩增和检测步骤准确性的关键工具。这种内参扩增对照适用于对不同标本中不同病毒载量的HSV进行早期可靠诊断。
