MOLgen

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"MOLgen DNA HHV-6 S1 Kit" is an assay kit for the detection of Human herpes virus (HHV) type 6 DNA by real-time PCR method.

Pathogen information: Herpesvirus-6 (HHV-6) is a member of the Herpesviridae family. These viruses contain DNA surrounded by a lipid envelope. HHV-6 exists as 2 closely related variants, HHV-6 A and HHV-6 B. HHV-6 transmission occurs most frequently through the shedding of viral particles into saliva. The salivary glands are regarded as an in vivo reservoir for HHV-6. The virus infects the salivary glands, establishes latency, and periodically reactivates to spread infection to other hosts [1]. The cases of vertical transmission has also been described. The infection may occur parenterally, i.e. during transplantations or blood transfusions.

Following the primary infection (which mainly occurs early in childhood), the virus establishes a latent infection in lymphocytes and monocytes and may persist in various tissues (blood, brain, liver, salivary glands, lungs, heart tissue, endothelium) with a low level of replication [2]. Mostadults (80%-90%) havebeeninfectedwiththisvirus.Primary infections in adults are rare.

The majority of HHV-6 infections are asymptomatic. The manifestations in immunocompetent people include fever that may exceed 40⁰C (persisting for 3-4 days) and exanthem subitum (roseola infantum), resembling measles or rubella symptoms. In immunocompromised patients, primary infection or reactivation may lead to serious complications. Clinical symptoms associated with primary or reactivated HHV-6 in adults may involve febrile illness, pneumonitis, chronic or fulminant hepatitis, meningoencephalitis, meningitis, and mononucleosis-like disease. Some acute graft-versus-host disease cases may be linked with HHV-6 reactivation [3]. Generally, variant B has been associated with exanthem subitum, whereas variant A has been found in immunosuppressed patients [4]. HHV-6 infection was also linked with other diseases such as multiple sclerosis andchronic fatigue syndrome.

Due to alteration of host immune response, HHV-6 can promote the development of opportunistic bacterial, fungal or viral infections. HHV-6 directly enhances replication of some viruses including other members of herpesviruses family such as EBV, CMV and HHV-8. HHV-6 can be a co-factor in progressing AIDS both through direct activation of HIV LTR by HHV-6 proteins and indirectly by HHV-6-induced suppression of host immune response, alteration of cytokine level and by up-regulation of CD4 expression in lymphocytes and extending the range of cellssusceptible to HIV-1 infection [5].

Because of the self-limiting nature of primary HHV-6 infection, laboratory diagnosis is rarely required in immunocompetent patients. Most often, the diagnosis is based on the clinical features. Diagnoses in patients who are recipients of organ transplants or patients with immunodeficiency, encephalitis, or hepatitis are performed by different laboratory methods. HHV-6 infection may be diagnosed by means of viral culture, serologic testing, or polymerase chain reaction (PCR) assay [6]. 

Rapid diagnosis of HHV-6 primary infections or reactivations can be facilitated by using quantitative PCR assays. Detection of co-infections with multiple herpesviruses can also be accomplished, with quantitative results enabling monitoring of virus load during antiviral therapy. As the virus spreads directly from cell to cell, the detection of viral DNA in acellular biofluids (e.g. serum, plasma, urine, saliva) helps to distinguish active from latent infection.

PCR analysis of HHV-6 DNA in cerebrospinal fluid should be used as a method of choice if CNS disorders are suspected. Detection of HHV-6 DNA by PCR can be used in case of uveitis for differential diagnostics of herpesvirus-induced ocular tissue damage or other viral infections [7].

Unlike other human herpesviruses, HHV-6 infection can lead to integration of the viral genome in human chromosomes, including the possibility that HHV-6 genome integrates in germline cells and can be transmitted to descendants.Individuals with inherited chromosomally-integrated HHV-6 have one copy of the viral genome integrated into the chromosome of every nucleated cell. Chromosomally integrated HHV-6 individuals will always have a very high viral load in whole blood testing - generally over 1 million copies per mL - but testing on plasma will be low or below the level of detection unless the individual is acutely ill [8].

Detection of HHV-6 DNA by PCR may be useful for:

differential diagnosis of infections in children with fever and rash;

• diagnosis of infectious mononucleosis in patients with negative Epstein-Barr virus tests;

• diagnosis of lymphoproliferative disorders;

• survey of organ or tissue recipients before and after transplantation;

• diagnosis of virus-associated diseases in patients with AIDS and other immunodeficiency states;

• control of antiviral treatment efficiency

“MOLgen DNA HHV-6 S1 Kit” is designed to detect HHV-6 DNA isolated from clinical specimens using extraction kit: “MOLgen Universal Extraction Kit”. An additional step of sample processing can be managed using the kit “MOLgen Hemolytic” before DNA isolation if whole blood specimens are concerned.

When using NA extraction kits of other manufacturers it is highly recommended to use internal control sample (IC) manufactured by Adaltis.

The assay is based on real-time polymerase chain reaction (PCR) method with fluorescent detection of amplified product. The kit detects the conserved HHV-6 DNA fragment.

The kit is designed for use with block cyclers iQ5 iCycler, CFX96 (Bio-Rad, USA), DT-96 (DNA-Technology Research and Production Company ZAO, Russia).

The kit contains reagents requiredfor 48 tests, including control samples.

“MOLgen DNA HHV-6 S1 Kit” is designed for the analysis of clinical materials (whole blood, blood serum, plasma, peripheral blood mononuclear cells, oral swabs, saliva, liquor, bone marrow, biopsy material).

Real time PCR is based on the detection of the fluorescence, produced by a reporter molecule, which increases as the reaction proceeds. Reporter molecule is dual-labeled DNA-probe, which specifically binds to the target region of pathogen DNA. Fluorescent signal increases due to the fluorescent dye and quencher separating by Taq DNA-polymerase exonuclease activity during amplification. PCR process consists of repeated cycles: temperature denaturation of DNA, primer annealing and complementary chain synthesis.

Threshold cycle value - Ct – is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal rises significantly above the background fluorescence. Increased fluorescence signal is due to the use of a specific for given DNA sequence DNA hybridization probe that in the course of reaction binds with one of the DNA strands, also providing additional specificity of the method. DNA probe comprises of a fluorescent dye at the 5’ end and of fluorescence quencher at the 3’ end which significantly reduces the fluorescence intensity. During the polymerase synthesis of the complementary strand, due to the 5’-3’ nuclease activity of Taq DNA polymerase the probe is cleaved from the 5’-terminus and separation of the quencher and the dye occurs, resulting in the increase the fluorescence signal due to accumulation of the reaction product. Fluorescence intensity detected depends on initial quantity of pathogen DNA template in the sample.

The use of Internal Control (IC) prevents generation of false negative results associated with possible loss of DNA template during specimen preparation. IC indicates if PCR inhibitors occur in the reaction mixture. IC template should be added in each single sample (including control samples) prior to DNA extraction procedure. The amplification and detection of IC does not influence the sensitivity or specificity of the target DNA PCR.