Real time PCR; Applications in diagnostics and research

ln recent years, real-time PCR has come forward as a robust and widely used molecular technique in clinical and biological. Although it can detect very minute quantities of target nucleic acid, but quantification of specific nucleic acids is not an easy task. Accurate and precise quantification is hampered by a number of factors that may include assay development and validation, fluorophores selection, handling during sample preparation, storage, reaction procedures, and batch analysis conditions. Even minor variations are significantly magnified by the exponential nature of this technique. Current review gives an insight of the advantages, limitations, assay chemistries, quantitation parameters, and quality control issues related to this technology. Moreover it will also highlight the utilization of Real time PCR in clinical oncology, virology, microbiology, and gene expression studies.

Predictors of response to antiviral therapy in patients with chronic hepatitis C from Pakistani population / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hepatitis C virus (HCV) constitutes a major public health issue around the world, especially in developing countries like Pakistan. In this study, we assessed outcome of interferon (INF) treatment in chronic hepatitis C patients categorized by gender, age, and viral load.</p><p><b>METHODS</b>In this study, 750 HCV positive patients with genotype 3 were selected, out of which 616 completed the entire treatment. Their personal history, pre-treatment HCV RNA and serum alanine transaminase (ALT) was quantified. Patients were treated with combination therapy of INF-α 2b three million units (thrice a week) plus ribavirin (1000 - 1200 mg per day) for 24 weeks. After 24 weeks their HCV RNA and serum ALT level was quantified.</p><p><b>RESULTS</b>Out of the 616 patients, 391 (63.5%) responded to therapeutic regimen (INF-α 2b plus ribavirin). Among the responders, 27.1% were men and 36.4% were women. Best treatment response was observed in patients having low viral load < 8 × 10(5) IU/ml and age ≤ 40 years than patients having low viral load and age > 40 years (73.2% vs. 60.3%, P = 0.05).</p><p><b>CONCLUSIONS</b>Better response to IFN-α 2b plus ribavirin was observed in patients with lower viral RNA and younger age. It suggests that all patients considered for treatment should have quantification of serum HCV RNA level. The result can be used to counsel patients on the likelihood of response and may influence the patient's decision on treatment.</p>

Mental retardation; molecular cytogenetic techniques used for diagnosis; a literature survey

Mental retardation, also termed as learning impairment or cognitive dysfunction, is a serious manifestation of nervous system. The defining features of mental retardation are low or subaverage intellectual functioning [Intelligence quotient<70], impairment in at least two of the adaptive skills [e.g communication ability, self care, self guidance, reading, writing ability, etc] before 18 year of age1. Molecular cytogenetics is the study of genetic disorders using advanced technologies combined with cytogenetic and molecular methodologies2. Molecular diagnosis has equal importance as clinical diagnosis in mental retardation and day by day new advancement in these methodologies are being introduced by molecular cytogeneticists. The promising achievement of molecular cytogenetic techniques is the genetic counseling of high risk pregnancies. The current mini-survey of literature discusses an overview of these techniques employed to investigate deletion, duplication, inversion and translocation of chromosomes associated with mental retardation

Significant incidence of typhoid in hepatitis C patients

Only proven way of transmission of Hepatitis C is through blood. The origin is unknown in nearly half the cases. Pollution is suspected as a cause but it is impossible to prove this relationship directly. We thought that typhoid being a proven pollution related disease, determination of its confection in Hepatitis C patients representing same Socio-economic group would be of interest. A typhoid in Hepatitis C patients can easily be overlooked because symptoms like fever and abdominal discomfort are present in both diseases. Blood samples were collected from three groups of study as mentioned in materials and methods. These samples were processed for 4[th] generation HCV ELISA. PCR for HCV, PCR for typhoid. Blood culture for typhoid and widal test as required [details are given in methodology]. Finally the data thus obtained was analysed and conclusions were drawn. Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering [NIBGE] and Millat Laboratory Faisalabad. April 2004 to Oct 2004. The parameters included were PCR, blood culture and widal test. There were three groups of study, PCR and ELISA positive patients of Hepatitis C [105] - further subdivided into two groups, with history of exposure to known causes of spread of HCV in last one year [65] and those without such history [40]; clinically diagnosed cases of typhoid [30]; and healthy controls [50]. In the three groups, PCR was positive in 9.5[7.7 and 12.5], 63.3, and 2.0% cases respectively. Figures for blood culture were 4.7[3.1 and 7.5], 33.3, and 0% in the same order, and the respective figures for widal test were 34.2[33.8 and 35.0], 56.6, and 24.0%. The increase in PCR and blood culture positivity in Hepatitis C cases as compared with normal subjects is statistically significant [P< 0.05]. These results clearly suggest that the source of infection for the two diseases is same in many cases, and therefore, provides a strong indication of a relationship between pollution and Hepatitis C