Molecular Diagnostic Unit
Qualitative and quantitative detection of HCV and HBV using Abbott Real Time PCR:
Hepatitis C virus (HCV) is a positive strand RNA virus, with a genome of 9,6 kb encoding a polyprotein that is post-translationally processed into structural and non structural proteins. HCV infection is characterized by chronicity and concerns of 170 millions of individuals worldwide. Chronically infected pateints present with liver injury, metabolic disorders and have a high risk of developming cirrhosis and hepatoceullar carcinoma.
Hepatits B virus (HBV) is a double stranded DNA virus composed of 27 nm nucleocapsid core (HBcAg) surrounded by an outer lipoportein coat containing the surface antigen (HBsAg). HBV is a prevalent infectious disease throughout the world. The clinical presentation of HBV ranges from subclinical hepatitis to symptomatic and chronic hepatitis which may evolve into cirrhosis and hepatocellular cancer.
Accurate measurement has been facilitated by the recent development of Capillary Electrophoresis (CE) using the FDA approved & first choice of College of American Pathologists (CAP) Sebia CAPILLARYSTM2. Which provides the precision, accuracy, linearity and correlation with an automated HPLC system that gives an excellent alternative to HPLC techniques in detection of thalassemias and hemoglobinopathies in addition to its sensitive detection of any minor abnormalities at the beginning of the disease shown by protein electrophoresis?
Alfa Lab now uses the Abbott Real Time PCR which is the most adavanced and senstive assay for detection of HCV and HBV.
The Abbott Real Time PCR has the following advantages:
- Sensitivity: 12 IU/ml for HCV and 10 IU/ml for HBV.
- Specificty: > 99.5% for HCV and HBV.
- The use of an internal control which is added during extraction and detected at all levels.
- Linear range from 12 IU/ml to 100 million IU/ml for HCV and from 10 IU/ml to 1 billion IU/ml for HBV.
Qualtitative and quantitative detection of CMV using Roche Lightcycler Real Time PCR:
Cytomegalovirus (CMV) belongs to the herbes virus family and infects more than 50% of the population worldwide. CMV infection is usually asymptomatic and often leads to undetected latent infection and re-infection. However, among immunocompromized patients such as AIDS, cancer patients and receipeints of transplant organs, CMV infection is a significant cause of disease and mortality. Quantitative Real Time PCR is an important tool for a pre-emptive disease mangement strategy i.e. early diagnosis, choice of relevant treatment and continued monitoring for the presence of invasive infections in pateints at risk of developing the disease.
LightMix for the detection of Mycobacterium spp/M. tuberculosis:
For the first time in Alfa Lab, the LightMix assay is being used which provides a fast, easy and accurate system to identify and quantify DNA from Mycobacterium tuberculosis along with other Mycobacterium species (M. bovis, M. africanum, and M. microti). This assay is very helpful for prognosis of the disease and evaluation of treatment .
Qualtitative and quantitative detection of BCR-ABL using Roche Lightcycler t(9;22) Quantification Kit:
The reciprocal translocation between the long arm of chromosome 9 and chromosome 22 can be detected by a number of techniques including flourescent in situ hyberdization (FISH), southern blotting, western blotting and reverse transcription polymerase chain reaction (RT-PCR).
Quantitative RT-PCR approaches allow the quantification of the transcript by competitive RT-PCR. The use of Lightcycler t(9;22) quantificatin kit has the following advantages:
- The lowest detection limit is one tumour cell.
- The assay determines the relative BCR-ABL expression levels by comparing them to the expression levels of a housekeeping gene (G6PDH).
- A positive control is contained in the kit.
Detection of the three most common mutations associated with the increased risk of thrombophilia:
These mutations are R506Q in Factor V gene, G20210A in Prothrombin gene and C677T in MTHFR gene. The FV/Proth/MTHFR StripAssay, used for the detection of these mutations, is based on the reverse hyberdization procedures.
Familial Mediterranean Fever (FMF)
Familial Mediterranean Fever (FMF) is an autosomal recessive inherited inflammatory disorder that is characterized by recurrent, short, self-limiting bouts of fever accompanied by pain in the abdomen, chest or joints. The most severe complication is progressive amyloidosis of the kidney that ultimately leads to renal failure. FMF is caused by mutations in the MEFV gene which codes for pyrin protein. Owing to the nonspecific clinical symptoms, molecular diagnosis significantly improves early and correct diagnosis of FMF, and allows to commence lifelong prophylactic treatment of affected individuals with colchicine. The FMF Strip Assay is based on the reverse hyberdization procedures which allow the detection of the twelve most common mutations causing FMF. FMF gene is now available at Alfa Laboratories.
Cardiovascular Disease (CVD)
Cardiovascular Disease (CVD) is a complex medical condition and the leading cause of death and disability in industerialized countries. The two major manifestations, atherosclerosis and venous thrombosis, are known to be caused by a complex interaction of multiple environmental and genetic parameters. Although separate pathophysiological entities, the two disorders are interrelated and share common risk factors, including advanced age, obesity, dislipidemia and diabetes.
The pathological process leading to atherosclerosis is characterized by endothelia dysfunction, hyperlipidemia, inflammation and hypertension. Polymorphisms in genes involved in these processes in combination with an unhealthy lifestyle (e.g. smoking, high-fat diet, physical inactivity) support thromboembolic events on disrupted atherosclerotic lesions in the vessel wall, the basic mechanism underlying myocardial infraction and ischemic stroke. Most gene variation contribute with minor effects, and the individual cardiovascular risk is related to a critical accumulation of detrimental polymorphisms acting in synergy with unfavourable environmental factors.
The CVD Strip Assay is based on the reverse hyberdization procedures for the detection of 17 mutations and polymorphisms in 13 genes :
- FV leiden [G1691A,R506Q],- FV R2 [H1299R],
- prothrombin G20210A,
- MTHFR C677T, - MTHRF A1298C,
- Factor XIII V34L,
- [PAI-1,serpin E1] 4G/5G,
- EPCR 4600 A>G,
- Apo B R3500Q, Apo E E2/E3/E4, FGB -455G>A.
- predisposing to atherossclerosis and venous thromboembolism.
Tests Performed in the Molecular Diagnostic Unit At Alfa Laboratories.
The CVD Strip Assay is now available at Alfa Laboratories.