Comparison of the polymerase chain reaction and Southern blot analysis in detecting and typing human papilloma virus deoxyribonucleic acid in tumors of the lower female genital tract

B. J. Monk, N. Cook, C. Ahn, S. A. Vasilev, M. L. Berman, S. P. Wilczynski

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

To conduct studies on the clinical and pathologic significance of human papilloma virus (HPV) in genital malignancies, accurate detection and typing of the virus in clinical material are essential. Currently, Southern blotting and the polymerase chain reaction (PCR) are two of the most commonly used methods to identify HPV. This study was undertaken to compare these techniques in the detection and typing of HPV in 242 invasive malignancies of the lower female genital tract. BamHI and PstI restriction digests of tumor DNA were hybridized to 32P-labeled probes for HPV types 6, 16, and 18 at Tm -20°C after Southern transfer. Blots were then washed at Tm -20°C and Tm - 9°C. The DNA was also amplified by PCR using both highly conserved consensus L1 primers that detect 25 different HPV genotypes and primers specific for HPV 6 E6, 16 E7, and 18 E6. All PCR products were hybridized to type-specific radiolabeled probes. In 202 of the 242 (83%) samples, HPV was detected, including 189 of 218 (87%) cervical cancers, 11 of the 20 (55%) vulvar cancers, and two of four tumors from the vagina, urethra, or anus. In 67% of the specimens, there was agreement between the Southern blot technique and both methods of PCR (consensus and type-specific primers), including 121 of the 202 HPV-positive specimens and 40 HPV-negative specimens. Of the 141 tumors with HPV detected by Southern blot analysis, the same HPV type was detected by PCR in 121 (86%). The discrepant typing for HPV in the tumors positive by Southern blot included four tumors negative by PCR, 10 tumors with a mixture of HPV types by PCR, five tumors positive by PCR with the consensus primers but negative with the corresponding type-specific primers; and, in one case, the Southern blot was read as HPV 16, whereas PCR showed the tumor to contain HPV 18. Of the 101 cases negative for HPV by Southern blot, HPV was detected in 61 (60%) cases by PCR. Forty-two cases were positive with both sets of primers, 15 cases were positive only with the consensus primers, and four cases were positive only with type-specific primers. Comparison of PCR detection of HPV by consensus and type-specific primers showed that 191 cases were positive with the consensus primers, 170 with the type-specific primers, and 163 positive using both sets of primers. In conclusion, PCR was more sensitive for detection of HPV in tumor tissue than Southern blot analysis, and consensus primers detected more HPV types than type-specific primers. There was good agreement between typing HPV by Southern blot and PCR when the Southern blot was positive. We concluded that PCR using both consensus and type-specific primers is optimal for detection of HPV in genital tumors.

Original languageEnglish (US)
Pages (from-to)283-291
Number of pages9
JournalDiagnostic Molecular Pathology
Volume3
Issue number4
StatePublished - 1994

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Papillomaviridae
Southern Blotting
Polymerase Chain Reaction
DNA
Neoplasms

Keywords

  • Genital cancers
  • HPV typing
  • PCR
  • Southern blot

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Comparison of the polymerase chain reaction and Southern blot analysis in detecting and typing human papilloma virus deoxyribonucleic acid in tumors of the lower female genital tract. / Monk, B. J.; Cook, N.; Ahn, C.; Vasilev, S. A.; Berman, M. L.; Wilczynski, S. P.

In: Diagnostic Molecular Pathology, Vol. 3, No. 4, 1994, p. 283-291.

Research output: Contribution to journalArticle

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abstract = "To conduct studies on the clinical and pathologic significance of human papilloma virus (HPV) in genital malignancies, accurate detection and typing of the virus in clinical material are essential. Currently, Southern blotting and the polymerase chain reaction (PCR) are two of the most commonly used methods to identify HPV. This study was undertaken to compare these techniques in the detection and typing of HPV in 242 invasive malignancies of the lower female genital tract. BamHI and PstI restriction digests of tumor DNA were hybridized to 32P-labeled probes for HPV types 6, 16, and 18 at Tm -20°C after Southern transfer. Blots were then washed at Tm -20°C and Tm - 9°C. The DNA was also amplified by PCR using both highly conserved consensus L1 primers that detect 25 different HPV genotypes and primers specific for HPV 6 E6, 16 E7, and 18 E6. All PCR products were hybridized to type-specific radiolabeled probes. In 202 of the 242 (83{\%}) samples, HPV was detected, including 189 of 218 (87{\%}) cervical cancers, 11 of the 20 (55{\%}) vulvar cancers, and two of four tumors from the vagina, urethra, or anus. In 67{\%} of the specimens, there was agreement between the Southern blot technique and both methods of PCR (consensus and type-specific primers), including 121 of the 202 HPV-positive specimens and 40 HPV-negative specimens. Of the 141 tumors with HPV detected by Southern blot analysis, the same HPV type was detected by PCR in 121 (86{\%}). The discrepant typing for HPV in the tumors positive by Southern blot included four tumors negative by PCR, 10 tumors with a mixture of HPV types by PCR, five tumors positive by PCR with the consensus primers but negative with the corresponding type-specific primers; and, in one case, the Southern blot was read as HPV 16, whereas PCR showed the tumor to contain HPV 18. Of the 101 cases negative for HPV by Southern blot, HPV was detected in 61 (60{\%}) cases by PCR. Forty-two cases were positive with both sets of primers, 15 cases were positive only with the consensus primers, and four cases were positive only with type-specific primers. Comparison of PCR detection of HPV by consensus and type-specific primers showed that 191 cases were positive with the consensus primers, 170 with the type-specific primers, and 163 positive using both sets of primers. In conclusion, PCR was more sensitive for detection of HPV in tumor tissue than Southern blot analysis, and consensus primers detected more HPV types than type-specific primers. There was good agreement between typing HPV by Southern blot and PCR when the Southern blot was positive. We concluded that PCR using both consensus and type-specific primers is optimal for detection of HPV in genital tumors.",
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