HIV-1 Detect™ Real Time PCR Kit

I. Description

The HIV-1 Detect™ Real Time PCR Kit is a ready-to-use kit containing all of the PCR reagents (except PCR master mix) needed for running quantitative, real-time DNA detection assays, including quantitative PCR, in the hydrolysis probe detection format. The HIV-1 Detect™ Real Time PCR Kit can be used to detect the presence of low levels of HIV-1 Proviral DNA. The kit can also be combined with reverse transcription to amplify cDNA from RNA samples for HIV-1 DNA detection. The HIV-1 Detect™ HIV-1 primers amplify a conserved 100 base pair region of the HIV-1 genome encoding the Gag protein. The presence of RT-PCR products is measured by detection of the fluorescence emitting FAM- labeled probe.

The unique features of the HIV-1 Detect™ Real Time PCR Kit include:

• Highly accurate, specific quantification of HIV from whole blood samples
• High sensitivity (detect less than 30 copies/well of target template)
• Wide linear dynamic range and good reproducibility
• Robust, accessible format, economical

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Background

The World Health Organization (WHO) estimates that as of 2007, 2 million children (age 15 and under) are living with HIV (1). WHO guidelines state that all infants younger than 1 year of age receive antiretroviral therapy (ART) (2). The importance of early identification of HIV infection is reflected in the 40% mortality rate within the first 12 months of perinatally infected infants that do not receive ART. In a randomized study of perinatally infected infants, ART initiated prior to 2 months of age reduced mortality by 76%, supporting the efficacy of early ART (3).

Early detection of HIV infection among infants has been difficult for much of the world population. In low and middle-income countries, only 8% of infants with HIV positive mothers are tested for HIV infection in the first 2 months (1). The use of serological assays to detect HIV in infants is unreliable as maternal HIV antibodies can remain in a child’s system for up to 18 months after birth. In contrast, detection of HIV is possible directly following the birth of a child by utilizing PCR to amplify HIV DNA or RNA from blood samples. Samples of blood collected on filter paper, or dried blood spots (DBS) from a heel stick provide a fast, efficient, and low cost means to transport samples for HIV detection. As opposed to alternate diagnostics, PCR based HIV detection requires only a very small amount of blood, as low as 50 µl. DBS are easy to prepare, can be stored at room temperature and represent minimal risk of infection to those handling samples during processing. Additionally, DBS are stable for long periods, providing a low-cost, efficient means to collect and transport samples from remote locations to laboratories (4).

The HIV-1 Detect™ Real Time PCR Kit is designed to assay the presence of HIV-1 Proviral DNA obtained by extraction of DNA from DBS samples. A small amount of blood (approximately 50 µl) collected on filter paper is sufficient to accurately assess the presence of absence of HIV-1 Proviral DNA in the circulatory system of potentially infected individuals.
The HIV-1 Detect™ Real Time PCR Kit can also be combined with reverse transcription to amplify cDNA from RNA samples for HIV-1 DNA detection.

II. Product Components and Storage Conditions

Kit Contents:

This kit contains reagents for approximately one plate of 96 reactions.
HIV-1 10X concentrated Probe and Primer Mix (green-capped vial)
HIV_1 positive control template dilution 1 (orange-capped vial)
Labeled tubes for preparation of HIV-1 positive control template dilution 2, 3, 4 and 5 (orange-capped vials)
HIV-1 negative control (purple-capped vial)
HIV_1 RNase/DNase free water (red-capped vials)

Storage and Stability:

If stored at –15 to –25°C, the product is stable through the expiration date printed on the label. For short-term storage (up to 3 months) the product may be stored at +2 to +8°C. The HIV-1 Detect™ Real Time PCR Kit components are light sensitive and should be kept away from the light. Avoid repeated freeze/thaw cycles and do not vortex the Probe and Primer mix.

Additional Equipment and Reagents Required:
Additional equipment and reagents required to perform real-time PCR assays with Custom HIV_1 DNA Probe and Primer mix, but not provided, include:
– DNA Extraction and Purification Kit
– Nuclease-free, aerosol-resistant tips
– Vortex mixer
– Micropipettes
– Sterile reaction tubes for preparing PCR mixes and dilutions
– Standard bench top micro centrifuge
– PCR reaction vessels (e.g., optical tubes or micro plates)
– Consumer Genetics PCR Master Mix or Applied Biosystems TaqMan® Universal PCR Master Mix
– Water, PCR grade
– Real time PCR instrument

III. Protocol

A. DNA Sample Preparation

DNA extraction kits are available from various manufacturers like QIAamp Viral DNA mini extraction kit or Bioneer Genomic DNA extraction kit. If dry blood spot (DBS) will be used for sample collection then the Detect™ DNA extraction and purification kit is recommended.

B. Prepare Reactions (HIV-1 Proviral DNA detection)

For each 10 µl reaction, prepare the following reaction mix:
1) Thaw the solutions and, for maximum recovery of the contents, briefly spin vials in a micro centrifuge before opening.
2) In a 1.5 ml reaction tube, prepare the PCR mix for one 10 µl reaction by adding the following components:

3) Pipette 5 µl PCR mix into each PCR reaction vessel or well of a PCR micro plate.
4) Add 5 µl of template or negative control DNA. To generate a standard curve use the included positive control template in triplicate.

C. PCR

1) Following the Operator’s Manual of your instrument supplier, program the instrument with the following parameters:

2) Place tubes or micro plate in the instrument and start the reaction.
3) At the end of the reaction, follow instrument instructions for quantification/analysis.

IV. Dynamic Range of Test

Under optimal PCR conditions, the HIV-1 Detect™ Real Time PCR Kit has a very high priming efficiency of >90% and can detect less than 30 copies of target template.

References

1) Towards Universal Access: Scaling up Priority HIV/AIDS interventions in the Heath Sector; Progress Report 2009. Geneva, Switzerland: World Heath Organization; 2009.
2) Anitretroviral Therapy for HIV Infection in Infants and Children: Towards Universal Access: Recommendations for a Public Health Approach. Geneva, Switzerland: World Health Organization; 2006.
3) Violari, A., Paed., F.C., Cotton, M.F., Med, M., Gibb., D.M., Babiker, A.G., Steyn, J., Madhi, S.A., Paed, F.C., Jean-Philippe, P., McIntyre, J.A.. (2008). Early Antiretroviral Therapy and Mortality among HIV-Infected Infants. N. Engl. J. Med 359; 2233-2244.
4) Lofgren, S.M., Morrissey, A.B., Chevallier, C.C., Malabeja, A.L., Edmonds, S., and Amos, B., von Seidlein, L., Schimana W., Stevens, W.S., Bartlett, J.A., Crump, J.A.. (2009). Evaluation of a dried blood spot HIV-1 RNA program for early infant diagnosis and viral load monitoring at rural and remote healthcare facilities. AIDS 2009; 23:2459-2466.

Note: The HIV & HBV PCR Kits are not available for Sale in the U.S at this moment!