Hybrid Capture 2 HPV DNA Test

 Hybrid capture 1 (HC1) was the original assay in a tube-based format, was approved in 1995. HC2 is a microtiter format-based test, developed by Digene Corporation, marketed presently by Quigen, got the FDA approval in 1999 and replaced HC1. The test was approved in 2003 for co-testing with routine cytology testing for women over age 30.

Steps and principle of the assay:

 • DNA is extracted and denatured from the sample.

 • A multigene RNA probe specific for high risk HPV16, –18, –31, –33, –35, –39, –45, –51,–52, –56, –58, –59, and –68 is added to it.

• If high-risk HPV DNA is present in the sample, DNA–RNA hybrids are formed.

• These are captured onto the well of the microtiter plate coated with monoclonal antibodies to DNA–RNA hybrids.

• A second monoclonal antibody conjugated to alkaline phosphatase is added.

• This binds to the captured hybrids in multiples.

• Light is produced. (The alkaline phosphatase dephosphorylates a chemiluminescent substrate which produces light. As the alkaline phosphatase acts on multiple copies of substrate, it generates an amplified signal for the presence of the target.)

Interpretation of the assay: The emitted light is measured in relative light units (RLU) on a luminometer.

One relative light unit (RLU) is considered as the cut-off value.

An RLU ³ cut-off value indicates the presence of high-risk HPV DNA but does not distinguish or specify the presence of specific HPV genotype.

An RLU < cut-off value indicates the absence of high-risk HPV DNA or high-risk HPV DNA below the limit of detection of the test.

Sensitivity: The detection limit of the test is 0.2 to 1 pg/ml, equivalent to 1,000 to 5,000 genome copies of HPV. False positivity and false negativity of 5% and 5–12% have been reported, respectively.

Cervista HPV HR test: This test detects 14 high risk HPV types including 13 that are detected by HC2 test and HPV 66 using sequence specific probes targeting the L1, E6, and E7 genes. It does not identify individual HPV type. It is based on the signal amplification chemistry. The test is FDA approved.

The detection limit of the test varies according to the HPV type:

• HPV16, –18, –31, –45, –52, –56: 1,250 to 2,500 copies per reaction,

• HPV33, –39, –51, –58, –59, –66, –68: 2,500 to 5,000 copies per reaction,

• HPV35: 5,000 to 7,500 copies per reaction.

Cobas 4800 HPV test: This test is a multiplex real-time PCR and nucleic acid hybridization test. It detects the L1 gene of 14 high-risk HPV types. Four fluorescent reporter probes are used which detect HPV16 and HPV18 individually and HPV –31, –33, –35, –39, –45,–51, –52, –56, –58, –59, and –68 as a pooled result.

The analytical sensitivity of different HPV types at the clinical cut-off is:

HPV45: 150 copies/mL; HPV31, –33, –39,–51, –59: 300 copies/mL, for HPV16, –18, –35, and –58: 600 copies/mL, and HPV56, –66, and–65:1,200 copies/mL, and 2,400 copies/mL for HPV52.

HPV mRNA PCR

This assay targets the detection of HPV E6/ E7 oncogene mRNA. During transient infection, the level of E6/E7 expression is less. In persistent infection, there is overexpression of E6/E7 mRNA. Detection of E6/E7 mRNA, therefore, reflects persistent infection and disease progression. Aptima assay is the FDA approved test for detection of HPV E6/E7 mRNA. Complementary oligomers are used for isolation of mRNA which is subjected to transcription mediated isothermal amplification (TMA) followed by hybridization protection assay using chemiluminescence for detection of amplified products.

Clinical implication of HPV DNA testing:

The detection of HPV DNA is important in case of cervical cancer screening for premalignant lesions. Cervical cancer screening is done by cytological screening with Pap smear examination.

Most of the major guidelines all over the world recommend the starting age for cervical cancer screening by cytology at 21 years. Screening by HPV DNA detection has been recommended from 25–30 years age as most of the women suffer from transient infection before this age, thereby positivity during that age has no clinical importance. Until 2014, most of the organizations were recommending the testing of HPV DNA along with cytology in women above 30 years of age. In April 2014, modified label of hrHPV test (high-risk HPV) was approved by Food and Drug Administration (FDA) for primary hrHPV testing in women above 25 years age. However, primary screening with hrHPV testing is not yet incorporated in clinical practice guidelines. Flowchart 4.1 depicts the cervical cancer screening guideline as recommended by major organizations along with the interim guidance regarding primary HPV DNA testing.

Advantages of HPV DNA testing: HPV DNA testing has shown higher sensitivity in detecting cervical intraepithelial neoplasia 2 and 3 (CIN 2, CIN3) as compared to that of cytology alone. In addition, negative HPV DNA test has more specificity and provides a higher level of reassurance than cytology.

اخر الاخبار

اخبار العتبة العباسية المقدسة

ضمن مجموعة مواكب أم البنين (عليها السلام) العتبة العباسية المقدسة تنهي استعداداتها لافتتاح الموكب الثقافي العام في طريق الزائرين

شعبة التوجيه الديني النسوي تُكمل استعداداتها لاستقبال زائرات الأربعين

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قسم الشؤون الفكرية يعقد اجتماعه الفصلي الثاني لعام 2025م

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الآخبار التكنلوجية

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مواضيع ذات صلة

platelet volume, mean (Mean platelet volume [MPV])

platelet count (Thrombocyte count)

platelet antibody detection (Antiplatelet antibody detection)

Future Directions of Molecular Diagnostic Testing

Next Generation Sequencing Technology

Analysis of Amplification Products

platelet aggregation test (Ristocetin test)

plasminogen activator inhibitor 1 (PAI-1)

phospholipase A2 receptor antibodies (anti-pla2r)

phosphate (PO4 , Phosphorus [P])

pheochromocytoma suppression and provocative testing (Clonidine suppression test [CST], Glucagon stimulation test)

parvovirus B19 antibody