title: Alteration in sample preparation to increase the yield of multiplex Polymerase Chain Reaction assay for diagnosis of genital ulcer disease.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Microbiology, National AIDS Research Institute, 73 G, MIDC, Bhosari, Pune-411 026, India.
authors.forename: G
authors.initials: G
authors.lastname: Rao
authors.forename: A
authors.initials: A
authors.lastname: Das
authors.forename: P
authors.initials: P
authors.lastname: Prabhakar
authors.forename: V
authors.initials: V
authors.lastname: Nema
authors.forename: A R
authors.initials: AR
authors.lastname: Risbud
mesh_terms: D005260:Female; D006801:Humans; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060737:Reproductive Tract Infections / Q000175:diagnosis* / Q000382:microbiology / Q000821:virology; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D014456:Ulcer / Q000175:diagnosis* / Q000382:microbiology / Q000821:virology