Development of In-House Multiplex Real Time PCR for Human Papillomavirus Genotyping in Iranian Women with Cervical Cancer and Cervical Intraepithelial Neoplasia

Abstract

Background: HPV related cervical cancer as one of the most common women cancers in developing countries. Regarding accessibility of commercial vaccines, any long or short term modality for integrating preventive immunization against HPV in a national program needs comprehensive information about HPV prevalence and its genotypes. The important role of selecting most accurate diagnostic technologies for obtaining relevant data is underlined by different assays proposed in the literature. The main objective of the present study was to introduce an in-house HPV typing assay using multiplex real time PCR with reliable results and affordable cost for molecular epidemiology surveys and diagnosis. Materials and Methods ResultsConclusions: According to acceptable performance, easy access to primers, probes and other consumables, affordable cost per test, this method can be used as a diagnostic assay in molecular laboratories and for further planning of cervical carcinoma prevention programs. Keywords Materials and Methods Patients' SpecimensEmbedded Tissue (FFPE) specimens) from women with known cervical precancerous and cancer referred to Tehran to meet ethical considerations, each patient was informed about the objectives of study and a signed a consent form before entering the study. After cytologic examination of collected Liquid Based Cytology (LBC) and FFPE blocks C and FFPE tissue blocks in a proper storage area until experimental phase. 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 containing partial length L1 sequence (20 e10 4 copy/ µl) were ® ,Germany, to be used for preparing standard samples necessary for evaluation studies. HPVs standard plasmidsExtraction of HPV DNA Viral DNA from Thin Prep preservatives and 20-30 mg Germany), according to the manufacturer's instructions. Design of primers and probesnucleotide). Analysis of extensive bioinformatics was done on primers and probes designed by Oligo7, Primer as internal control primers and probe were considered in inhibition and quality of extracted DNA. In-house multiplex real time PCR for HPV genotypingas quencher dye at the 3' end of probes sequences. For carried out in a 25µl reaction volume containing 12.5µl 2Xand 5 µl DNA extracted. These reaction mixtures were Orange Channels). Furthermore, all of the four mixtures were tested against known positive specimens of HBV, dilutions of HPV plasmids were prepared from 1 to 1000 copy/ µl as separated and mixed samples. ResultsThe quality and performance of developed assay was evaluated focusing on analytical sensitivity (detection negative and 20 HPV positive samples (both as single specimens and mixed cocktails with different copy numbers of each genotype per micro liter from 1 to 1000) signal in all samples.Analytical sensitivity high risks and low risks HPV genotypes) were tested and only one sample (genotype 52) was falsely negative.Detection limit prepared (from 1 to 1000 copy /µl) and the assay could detect 5 copy/µl of each HPV genotypes in single and mixed cocktails. Data for these evaluation...

Cervical Cancer and Genital Infections: Assessment of Performance and Validation in Human Papillomavirus Genotyping Assays in Iran, its Neighbouring Countries and Persian Gulf Area

“…Other used genotyping methods were unconfirmed protocols or did not fully cover all necessary types such as some PCR hybridization methods, PCR sequencing, multiplex PCR, and Real Time PCR method. Only one home-brew HPV genotyping method, In-House Multiplex Real time PCR, was approved in WHO proficiency testing among these reports ( 12 ). Overall, 13 out of 46 papers used protocols covering all necessary detected types consisting of 10 approved methods, 2 commercially unapproved procedures and one home brew developed method ( Table 1 and 2 ).…”

“…However, the most important objective is HPV diagnosis in early stages of cervical cancer and precancerous lesions and other related cancers and infections ( 67 - 71 ). Unfortunately, our search showed no special attention on the majority of home brew methods to optimize the test in applied conditions, regarding detection limit, frequency, and false negative and positive results, except a few studies ( 12 ). Another missed point in these papers was ignoring HPV multiple infections that were underlined in reports applying standard and approved methods ( 4 , 72 - 78 ).…”

Glutathione S-transferase omega gene polymorphism as biomarker for human papilloma virus and cervical cancer in iranian women

“…To the best of our knowledge, no other previous study has investigated this issue in Iranian populations. Cervical cancer is one of the more prevalent causes of death among women, leading to approximately 270,000 deaths annually ( 2 , 19 , 20 ). Persistent genital infections with different high-risk HPV genotypes, specifically HPV 16 and 18, lead to CIN and cervical cancer ( 1 , 21 , 22 , 23 ).…”

Characterization of JAK2 V617F (1849 G ＞ T) Mutation in Cervical Cancer Related to Human Papillomavirus and Sexually Transmitted Infections

“… 1 , 2 Persistent high risk HPV genotypes with other genital pathogens are essential for enhancing the susceptibility to genital malignancies. 3 – 7 …”