Indian Journal of Medical Microbiology IAMM  | About us |  Subscription |  e-Alerts  | Feedback |  Login   
  Print this page Email this page   Small font sizeDefault font sizeIncrease font size
 Home | Ahead of Print | Current Issue | Archives | Search | Instructions  
Users Online: 1296 Official Publication of Indian Association of Medical Microbiologists 
 ~   Next article
 ~   Previous article
 ~   Table of Contents

 ~   Similar in PUBMED
 ~  Search Pubmed for
 ~  Search in Google Scholar for
 ~Related articles
 ~   Citation Manager
 ~   Access Statistics
 ~   Reader Comments
 ~   Email Alert *
 ~   Add to My List *
 * Requires registration (Free)
 

 Article Access Statistics
    Viewed2167    
    Printed83    
    Emailed2    
    PDF Downloaded161    
    Comments [Add]    

Recommend this journal

 

 ORIGINAL ARTICLE
Year : 2013  |  Volume : 31  |  Issue : 1  |  Page : 15-18

Alteration in sample preparation to increase the yield of multiplex Polymerase Chain Reaction assay for diagnosis of genital ulcer disease


1 Department of Microbiology, National AIDS Research Institute, 73 'G', MIDC, Bhosari, Pune-411 026, India
2 Department of Microbiology, Family Health International, 360, New Delhi, India

Correspondence Address:
A R Risbud
Department of Microbiology, National AIDS Research Institute, 73 'G', MIDC, Bhosari, Pune-411 026
India
Login to access the Email id

Source of Support: Financial Assistance from Bill and Melinda Gates Foundation for STI- OR study among HRGs., Conflict of Interest: None


DOI: 10.4103/0255-0857.108709

Rights and Permissions

Purpose: Genital Ulcer Disease (GUD) is common sexually transmitted infection (STI). Multiple studies have shown that GUDs are strongly associated with the transmission and the acquisition of HIV infection. An accurate diagnosis of common etiology of GUD namely Herpes, syphilis and Chancroid is possible using Multiplex PCR (M-PCR). However, frequent presence of Polymerase Chain Reaction inhibitors in the ulcer swab specimen limits the performance of the assay. In order to overcome this problem, alternative specimen preparation method was used. Materials and Methods: To determine the common etiology, GUD specimens obtained under an STI operations research study were tested with M-PCR after the samples were prepared using Roche Amplicor specimen preparation kit. PCR inhibiting samples were identified from that, which showed negative results. These samples were subjected to phenol-chloroform extraction and ethanol precipitation before the conduct of M-PCR on them. Results: Of the 237 GUD specimens tested, in 145 etiologies could be detected, whereas 92 samples were found negative. Further spiking with one of the target DNA, 128 of the negative samples were found to contain the inhibitors. These 126 samples were then subjected to phenol chloroform extraction and ethanol precipitation followed by M-PCR. Using this method for sample preparation, etiology could be determined in 46 (23%) additional samples. This success rate of altered sample preparation method has been lower than that has reported. Conclusion: The results indicate that sample preparation using phenol chloroform extraction and ethanol precipitation, prior to M-PCR helps to eliminate the inhibitors and increase the yield of the assay. However, being a laborious procedure, it may be used for samples giving negative results after the screening by Roche Amplicor specimen preparation kit.






[FULL TEXT] [PDF]*


        
Print this article     Email this article

2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04