Increased sensitivity and specificity of Borrelia burgdorferi 16S ribosomal DNA detection.

The DNA of Borrelia burgdorferi spirochetes extracted by ammonium hydroxide was used as the template for nested polymerase chain reaction (PCR) amplification of the species-specific 16S ribosomal DNA (rDNA). The primers were those well known to be specific for signature sequence amplification of the B burgdorferi sensu lato 16S ribosomal RNA gene. The positive 293-base-pair nested PCR amplicon was subjected to routine direct automated Sanger sequencing.

Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens.

Aims: Persistent infection indicated by detection of human papillomavirus 16 (HPV-16) on repeat testing over a period of time poses the greatest cervical cancer risk. However, variants of HPV-16, HPV-31 and HPV-33 may share several short sequence homologies in the hypervariable L1 gene commonly targeted for HPV genotyping. The purpose of this study was to introduce a robust laboratory procedure to validate HPV-16 detected in clinical specimens, using the GenBank sequence database as the standard reference for genotyping.

Validation of human papillomavirus genotyping by signature DNA sequence analysis.

Background: Screening with combined cytologic and HPV testing has led to the highest number of excessive colposcopic referrals due to high false positive rates of the current HPV testing in the USA. How best to capitalize on the enhanced sensitivity of HPV DNA testing while minimizing false-positive results from its lower specificity is an important task for the clinical pathologists.

Methods: The HPV L1 gene DNA in liquid-based Pap cytology specimens was initially amplified by the degenerate MY09/MY11 PCR primers and then re-amplified by the nested GP5+/GP6+ primers, or the heminested GP6/MY11, heminested GP5/MY09 primers or their modified equivalent without sample purification or DNA extraction.

Molecular tests for human papillomavirus (HPV), Chlamydia trachomatis and Neisseria gonorrhoeae in liquid-based cytology specimen.

Background: Laboratory detection of Human papillomavirus (HPV), Chlamydia trachomatis and Neisseria gonorrhoeae in liquid-based cervicovaginal cytology specimens is now based on identification of the DNA sequences unique to these infectious agents. However, current commercial test kits rely on nucleotide probe hybridization to determine DNA sequences, which may lead to diagnostic errors due to cross-reactivity. The aim of this study was to find a practical approach to perform automated Sanger DNA sequencing in clinical laboratories for validation of the DNA tests for these three infectious agents.

HPV infection among women in a representative rural and suburban population of the USA.

Objective: To study the prevalence of low-risk and high-risk HPV genotypes in a largely suburban, non-Hispanic, white female population of the USA, and to determine the positive predictive value of one-occasion HPV detection and genotyping using high-grade squamous intraepithelial lesion (HSIL) cytology as the endpoint for clinical evaluation.

Methods: HPV DNA present in liquid-based cytology specimens collected by gynecologists in private practice was amplified using nested polymerase chain reaction. HPV DNA was validated by signature DNA sequencing for accurate genotyping.

DNA sequencing validation of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acid tests.

DNA sequencing was used to confirm Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids in endocervical swab samples. DNA in residues of the samples with positive results by 2 commercial kits was subjected to nested polymerase chain reaction (PCR) amplification. The nested PCR amplicons were used as templates for direct automated DNA sequencing.

Routine human papillomavirus genotyping by DNA sequencing in community hospital laboratories.

Background: Human papillomavirus (HPV) genotyping is important for following up patients with persistent HPV infection and for evaluation of prevention strategy for the individual patients to be immunized with type-specific HPV vaccines. The aim of this study was to optimize a robust "low-temperature" (LoTemp) PCR system to streamline the research protocols for HPV DNA nested PCR-amplification followed by genotyping with direct DNA sequencing. The protocol optimization facilitates transferring this molecular technology into clinical laboratory practice.