The genesig®PLEX qPCR Kit HBV/HCV/HIV1/HIV2 is designed for the in vitro detection of Human Hepatitis Viruses B and C and Human Immunodeficiency Viruses 1 & 2.
HIV1 & HIV2:Human immunodeficiency virus (commonly known as HIV) is a retrovirus that is the cause of the disease known as AIDS (Acquired Immunodeficiency Syndrome), a syndrome where the immune system begins to fail, leading to many life-threatening opportunistic infections
HIV is transmitted through direct contact of a mucous membrane with a bodily fluid containing HIV, such as blood, semen, vaginal fluid, preseminal fluid or breast milk. This transmission can come in the form of: penetrative (anal or vaginal) sex, oral sex, blood transfusion, contaminated needles, exchange between mother and infant during pregnancy, childbirth, or breastfeeding, or other exposure to one of the above bodily fluids. Two species of HIV infect humans: HIV-1 and HIV-2. HIV-1 is hypothesised to have originated in southern Cameroon after jumping from wild chimpanzees (Pan troglodytes troglodytes) to humans during the twentieth century. HIV-2 is hypothesised to have originated from the Sooty Mangabey (Cercocebus atys), an Old-World monkey of GuineaBissau, Gabon, and Cameroon. HIV-1 is more virulent, more easily transmitted and is the cause of the majority of HIV infections globally, while HIV-2 is less easily transmitted and is largely confined to West Africa.
HBV & HCV:Originally known as serum hepatitis, hepatitis B (HBV) has only been recognized as such since World War II and has caused current epidemics in parts of Asia and Africa. Hepatitis B is recognized as endemic in China and various other parts of Asia. Over one-third of the world's population has been or is actively infected by hepatitis B virus (HBV). The hepatitis B virus is a member of the Hepadnavirus family. It consists of a proteinaceous core particle containing the viral genome in the form of double stranded DNA with single-stranded regions and an outer lipid-based envelope with embedded proteins. The envelope proteins are involved in viral binding and release into susceptible cells. The hepatitis C virus (HCV) is a small, enveloped, single-stranded, positive sense RNA virus in the family Flaviviridae. HCV mainly replicates within hepatocytes in the liver, although there is clear evidence for replication in lymphocytes or monocytes. Circulating HCV particles bind to receptors on the surfaces of hepatocytes and subsequently enter the cells. Once inside the hepatocyte, HCV utilizes the intracellular machinery necessary to accomplish its own replication.
Sample Type:All kinds of sample material suited for PCR amplification can be used. Please ensurethe samples are suitable in terms of purity, concentration, and RNA/DNA integrity
Always run at least one negative control with the samples. To prepare a negative control,replace the template RNA sample with RNase/DNase free water.
Dynamic Range of Test:Under optimal PCR conditions genesig kits have very high priming efficiencies of >90%and can detect between 1X10e8 and 1X10e2 copies of target template.
Specificity:The genesigPLEX kit is designed for the in vitro detection of Human Immunodeficiency Viruses 1 & 2 and Human Hepatitis Viruses B and C
The primers and probe sequences in this kit have 100% homology with a broad range of clinically relevant reference sequences based on a comprehensive bioinformatics analysis. They therefore have a very broad detection profile.
Test Princple:Real-time PCR:Individual primer and probes designed for each virus have been combined into a single reaction and these can be detected through the different fluorescent channels as described in the kit contents. The primer and probe mix provided exploits the so-called TaqMan® principle. During PCR amplification, forward and reverse primers hybridize to the target cDNA. Fluorogenic probes are included in the same reaction mixture which consists of a DNA probe labelled with a 5`-dye and a 3`-quencher. During PCR amplification, the probe is cleaved, and the reporter dye and quencher are separated. The resulting increase in fluorescence can be detected on a range of qPCR platforms.
Positive Control:For a positive control, the kit contains a single positive control that contains templates for the 4 targets in the test. The kit positive control will give an HIV1 and HIV2 signal through the FAM channel, an HBV signal through the VIC channel and an HCV signal through the ROX channel. Each time the kit is used, at least one positive control reaction must be included in the run. A positive result indicates that the primers and probes for detectingeach virus are working properly in that particular run. If a negative result is obtained the test results are invalid and must be repeated. Care should be taken to ensure that the positive control does not contaminate any other kit component which would lead to false positive results. This can be achieved by handling these components in a post PCR environment. Care should also be taken to avoid cross-contamination of other samples when adding the positive control to the run. This can be avoided by sealing all other samples and negative controls before pipetting the positive control into the positive control well.
Endogenous Control:To confirm extraction of a valid biological template, the single primer/probe mix supplied contains primers and probe designed to detect an endogenous gene. Detection of the endogenous control is through the Cy5 channel. A poor endogenous control signal may indicate that the sample did not contain sufficient biological material.
Negative Control:To confirm the absence of contamination, a negative control reaction should be included every time the kit is used. For this reaction, the RNase/DNase free water should be used instead of template. A negative result indicates that the reagents have not become contaminated while setting up the run.
Kit Components:
Multiplex primer/probe mix (100 reactions BROWN)FAM, VIC, ROX and Cy5 labelled (see table)
Multiplex positive control template (RED)
Lyophilized oasig™ OneStep Master Mix (RED)
oasig™ resuspension buffer (BLUE)
Template preparation buffer (YELLOW)for resuspension of positive control template
RNase/DNase free water (WHITE)for resuspension of primer/probe mix
Reagents and Equipment to be supplied by the User:Real-time PCR InstrumentRNA extraction kitThis kit is recommended for use with genesig EASY DNA/RNA Extraction kit. However, it is designed to work well with all processes that yield high quality RNA with minimalPCR inhibitors
Pipettors and tipsVortex and centrifugeThin walled 1.5ml tubesqPCR plates
Storage and Stability:This kit is stable at room temperature but should be stored at -20ºC on arrival. Once the lyophilized components have been resuspended they should not be exposed to temperatures above -20ºC for longer than 30 minutes at a time and unnecessary repeated freeze/thawing should be avoided. The kit is stable for six months from the date of resuspension under these circumstances. Primerdesign does not recommend using the kit after the expiry date stated on the pack.
