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“Molgen DNA EBV S1 Kit” is an assay kit for the detection of Epstein-Barr Virus DNA by Real-Time PCR method.

Pathogen information EBV is a herpesvirus that infects the vast majority of the world's adult population. Comparatively large (172 kilobase pairs) EBV genome contains about 85 genes not only involved in direct viral replication but also helping to avoid host immune response and control cell cycle. Following primary infection, EBV persists in the infected host as a lifelong asymptomatic infection. Although EBV can infect different cell types, it is B-cells and epithelial cells in the oropharynx that maintain a major part of viral replication for a long period. Some of infected B-cells become memory cells where EBV stays latent indefinitely. Latent EBV in B-cells can be reactivated and switch to lytic replication [1].

The virus can be found in many types of bodily fluids of infected person but commonly is transmitted with saliva. Primary infection in childhood is usually asymptomatic or cause nonspecific mild respiratory disease. However, in western countries with affluent populations, where exposure is generally delayed until adolescence or young adulthood, primary infection is commonly associated with infectious mononucleosis (IM). Over 50 percent of patients with IM manifest the triad of fever, lymphadenopathy, and pharyngitis; splenomegaly, palatal petechiae, and hepatomegaly are each present in >10 percent of patients. Less common complications include hemolytic anemia, thrombocytopenia, aplastic anemia, myocarditis, hepatitis, genital ulcers, splenic rupture, rash, and neurologic complications such as Guillain–Barré syndrome, encephalitis, and meningitis [1]. Virus DNA persists at high levels in the saliva of IM patients as long as for 6 months after initial infection. IM patients remain highly infectious during convalescence [2]. Due to alteration of host immune response, EBV can contribute to invasion of bacterial, fungal or viral agents.

EBV may exist in both latent and lytic states. In contrast to other herpesviruses, it is in the latent state that the virus is generally associated with serious disease such as tumor development. EBV is present in approximately 95% of all African Burkitt's lymphomas. EBV is associated with lymphoproliferative disease in the immunocompromised people. In organ transplant recipients, B-cell lymphoproliferative disease is virtually always EBV-associated. Other tumors that are almost universally EBV-associated include nasopharyngeal carcinoma and lymphomatoid granulomatosis, whereas cancers such as acquired immune deficiency syndrome (AIDS)-related lymphoma and Hodgkin lymphoma harbor EBV in only about half of the cases. When a cancer is EBV-related, the viral DNA appears to be present in virtually every malignant cell, thus serving as a marker for the tumor clone [3, 4].

The diagnostic strategies differ between immunocompromised and immunocompetent individuals due to the distinct therapeutic interventions required. Because the time of intervention is a critical factor in immunocompromised patients, a diagnostic method must meet the following criteria: early detection of EBV replication and a high positive predictive value for the respective disease, thus enabling preemptive therapy. In addition, monitoring of therapy should be possible. Thus, direct detection methods, including PCR, mainly meet this profile. In immunocompetent individuals the key issue of EBV diagnostics is the detection or exclusion of a primary, past, or no EBV infection. Therefore, serology provides rational criteria for interpretation of the results and serological assays are preferred [5].

Plasma is a convenient sample type for PCR testing because EBV viral loads are usually very low or undetectable in plasma of healthy individuals, but they are increased during active EBV infection and in many EBV-related malignancies [4,6]. In contrast, EBV DNA in blood leukocytes or saliva can be detected for months after active infection [2]. Thereafter a whole blood or leukocyte fraction is generally used only for viral load measurement and their qualitative analysis is uninformative.

When central nervous system is affected, EBV DNA can be found in cerebrospinal fluid (CSF). EBV causes aseptic meningitis, encephalomyeloneuritis and neuritis. The EBV neuropathies are manifested by ophthalmoplegia, lumbosacral plexopathy, and sensory or autonomic neuropathy. However, EBV is latent in B-cells, and caution should be used in attributing neurological disease to EBV purely on the basis of a positive CSF PCR; in previously EBV-infected individuals, PCR might detect EBV DNA latent in B-cells that are part of the inflammatory response induced by another agent. The presence of anti-EBV IgM or IgG antibody in CSF is more likely to be significant although serological test can fail at an early stage of the disease. Thus, the best strategy for the survey of EBV-induced neurological disease is to use a combination of these methods [7].

EBV DNA can be detected in the aqueous humor and vitreous fluids in uveitis although positive PCR for EBV DNA does not exclude the presence of another causative viral agent such as varicella-zoster virus; viral load should be taken into account [8].

“Molgen DNA EBV S1 Kit” is designed for detection of EBV 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 highly conserved DNA fragment of the gene LPM2a (related to KC207814 DNA sequence) specific to all known EBV isolates.

The kit contains reagents requiredfor 96 tests, including control samples.

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

“Molgen DNA EBV S1 Kit”is designed for the analysis of clinical materials (whole blood, serum, plasma, cerebrospinal fluid, oropharynx swab, saliva, vitreous fluid, biopsy material).

When extracting EBV DNA from blood serum (plasma) using “MOLgen Universal Extraction Kit” and from whole blood using “MOLgen Universal Extraction Kit” and ”MOLgen Hemolytic” a quantitative determination of EBV DNA in the sample is possible.

When extracting EBV DNA from blood serum (plasma) using “MOLgen Universal Extraction Kit” a quantitative determination of EBV DNA in the sample is possible.

Attention! The use of:

• NA extraction kits from other manufacturers;

• other real-time PCR devices;

• reaction volumes, other than 50 μL have to be validated in the laboratory by the user. Special notes regarding the internal control (IC) have to be strongly followed.

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.