title: Evaluation of highly conservedhsp65-specific nested PCR primers for diagnosingMycobacterium tuberculosis.
abstract: To evaluate the sensitivity and specificity of a new nested set of primers designed for the detection of Mycobacterium tuberculosis complex targeting a highly conserved heat shock protein gene (hsp65).
  
  
  
  The nested primers were designed using multiple sequence alignment assuming the nucleotide sequence of the M. tuberculosis H37Rv hsp65 genome as base. Multidrug-resistant Mycobacterium species along with other non-mycobacterial and fungal species were included to evaluate the specificity of M. tuberculosis hsp65 gene-specific primers.
  
  
  
  The sensitivity of the primers was determined using serial 10-fold dilutions, and was 100% as shown by the bands in the case of M. tuberculosis complex. None of the other non M. tuberculosis complex bacterial and fungal species yielded any band on nested polymerase chain reaction (PCR). The first round of amplification could amplify 0.3 ng of the template DNA, while nested PCR could detect 0.3 pg.
  
  
  
  The present hsp65-specific primers have been observed to be sensitive, specific and cost-effective, without requiring interpretation of biochemical tests, real-time PCR, sequencing or high-performance liquid chromatography. These primer sets do not have the drawbacks associated with those protocols that target insertion sequence 6110, 16S rDNA, rpoB, recA and MPT 64.
authors.affiliation: Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
authors.forename: P
authors.initials: P
authors.lastname: Priyadarshini
authors.affiliation: Department of Microbiology, Bhopal Memorial Hospital and Research Centre, Bhopal, India.
authors.forename: K
authors.initials: K
authors.lastname: Tiwari
authors.affiliation: Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
authors.forename: A
authors.initials: A
authors.lastname: Das
authors.affiliation: Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
authors.forename: D
authors.initials: D
authors.lastname: Kumar
authors.affiliation: Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
authors.forename: M N
authors.initials: MN
authors.lastname: Mishra
authors.affiliation: Department of Microbiology, Bhopal Memorial Hospital and Research Centre, Bhopal, India.
authors.forename: P
authors.initials: P
authors.lastname: Desikan
authors.affiliation: Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
authors.forename: G
authors.initials: G
authors.lastname: Nath
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics*; D003362:Cost-Benefit Analysis; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D017931:DNA Primers; C074119:heat-shock protein 65, Mycobacterium