Application of a multiplex PCR to cervical cells collected by a paper smear for the simultaneous detection of all mucosal human papillomaviruses (HPVs) and typing of high-risk HPV types 16 and 18.
A simple paper smear (PS) method for dry collection and storage of cervical specimens was employed to develop an easy multiplex (MPX) PCR for simultaneous detection of generic human papillomaviruses (HPVs) as well as typing of the high-risk HPV-16 and -18, the two clinically most important HPV genotypes, which are responsible for more than 80 % of cervical cancers. Multiplexing was performed with a small amount of DNA eluted by boiling from a single PS punch in a single tube and using a mixture of four pairs of primers specific for the HPV L1 consensus sequence, HPV-16, HPV-18 and the β-globin gene. Sixty HPV-positive biopsies and corresponding PS specimens from cervical cancer patients as well as cervical smears from 100 healthy women with or without abnormal cytology were collected both as PSs and in PBS. Detection of HPV DNA from cervical biopsies collected in PBS and corresponding cervical scrapes on a PS or in PBS by conventional and MPX-PCR showed a concordance of 100 % and adequacy of 93 %. A similar comparative study in cervical scrapes from normal women also revealed 100 % concordance. The technique was validated in a multicentric study at four different national laboratories. PSs collected by different centres showed variable adequacy (73-82 %) but the use of multiple PS discs for DNA extraction significantly increased the adequacy. Integration of PSs with MPX-PCR for the detection and typing of HPVs is a highly convenient, efficient, simple and cost-effective method for large-scale clinico-epidemiological studies and is also suitable for HPV vaccine monitoring programmes in resource-poor settings.
Shukla
Shirish
S
Division of Molecular Oncology, Institute of Cytology & Preventive Oncology, Plot I7, Sector 39, Noida, India.
Bharti
Alok C
AC
Division of Molecular Oncology, Institute of Cytology & Preventive Oncology, Plot I7, Sector 39, Noida, India.
Mahata
Sutapa
S
Division of Molecular Oncology, Institute of Cytology & Preventive Oncology, Plot I7, Sector 39, Noida, India.
Hussain
Showket
S
Division of Molecular Oncology, Institute of Cytology & Preventive Oncology, Plot I7, Sector 39, Noida, India.
Hedau
Suresh
S
Division of Molecular Oncology, Institute of Cytology & Preventive Oncology, Plot I7, Sector 39, Noida, India.
Sharma
Rajyashri
R
Jawaharlal Nehru Medical College and Hospital, Aligarh, India.
Pillai
M Radhakrishna
MR
Rajiv Gandhi Centre for Biotechnology, Trivandrum, Kerala, India.
Krishna
Sudhir
S
National Centre for Biological Sciences, Bangalore, India.
Chiplunkar
Shubhada
S
Advanced Centre for Treatment and Research Education in Cancer, Mumbai, India.
Tongaonkar
Hemant
H
Tata Memorial Hospital, Mumbai, India.
Das
Bhudev C
BC
Dr B. R. Ambedakar Centre for Biomedical Research, University of Delhi, Delhi, India.|Division of Molecular Oncology, Institute of Cytology & Preventive Oncology, Plot I7, Sector 39, Noida, India.
D002584:Cervix Uteri / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D003890:Desiccation; D005260:Female; D052162:Human papillomavirus 16 / Q000235:genetics / Q000302:isolation & purification; D052161:Human papillomavirus 18 / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D014626:Vaginal Smears / Q000379:methods*; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D004279:DNA, Viral