Detection and Typing of HSV-1, HSV-2, CMV and EBV by Quadruplex PCR

The development of a multiplex polymerase chain reaction (PCR) method for rapid and accurate detection and typing of herpes simplex virus type 1 (HSV-1), and type-2 (HSV-2), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) is very important for clinical diagnosis to allow the deliver of therapy as early as possible. Large scale amplifications by multiplex PCR of viral DNA can lower the cost and time for viral diagnosis. In this study, therefore sensitive quadruplex PCR was achieved by optimizing parameters such as primers, and 1.5 mM magnesium and 200 uM dNTPs concentrations. The concentrations of HSV-1, HSV-2, CMV and EBV primers were 0.5, 0.3, 0.25 and 0.25 pmoles, respectively. Optimal annealing temperature was 54 degrees C. Employing these conditions, we could detect 10 copies of reconstructed template plasmid DNA, which were cloned to vectors containing target sequences of viral DNA. PCR products of 271 bp for HSV-1, 231 bp for HSV-2, 368 bp for CMV, and 326 bp for EBV were separated on 5.0% polyacrylamide gel electrophoresis and confirmed by direct sequencing. The present study showed that the quadruplex PCR assay described herein has potential application in clinical diagnosis, when rapid, accurate detection and typing of viruses HSV-1, HSV-2, CMV or EBV are necessary.

Polymerase chain reaction based restriction fragment length polymorphism for the genotyping of cytomegalovirus (CMV) from patients with CMV disease in Chennai.

BACKGROUND & OBJECTIVES: Since cytomegalovirus (CMV) is heterogenous and exhibits genomic polymorphism, polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) was applied to identify the glycoprotein B subtypes in patients diagnosed to have CMV disease. METHODS: CMV standard strain (AD 169) and 55 clinical specimens from 50 patients (35 males; 15 females) positive for CMV by PCR coding for the morphological transforming region II gene were subjected to PCR coding for gp 55 region of CMV gB gene. PCR amplified products were subjected to RFLP using Hinf I. RESULTS: Of 50 patients, 26 (52.0%) (20 males; 6 females) were positive by PCR (gB gene). Upon RFLP, AD 169 and CMV strains of 14 (53.8%) patients (14 males) were typed as subtype II, and 12 (46.1%) (6 males; 6 females) as subtype III. Of 11 paediatric patients, 7 (63.6%) were infected with CMV subtype II and 4 (36.4%) with subtype III. CMV strains of the dual specimens from 3 PCR positive patients belonged to the same subtype. INTERPRETATION & CONCLUSION: In this study on CMV genotypes, CMV gB subtypes II and III were found in 53.8 and 46.1 per cent patients studied respectively, while I and IV were not present.

Diagnóstico por cultivo de tejidos del citomegalovirus / Diagnosis by tissue culture of cytomegaloviruses

El citomegalovirus (CMV) infecta aproximadamente al 1% de los recién nacidos, siendo una de las principales causas de retraso mental y sordera. Además de ser un patógeno muy importante en pacientes inmunosuprimidos. El diagnóstico de laboratorio de infección por CMV será motivo de comentario en una serie de artículos, ya que es un problema de salud pública. En éste, se describen cómo prepara y mantener fibrosblastos de prepucio de recién nacido (HFF), cómo llevar a cabo el aislamiento del CMV a partir de especímenes clínicos, a través de las técnicas clásicas de cultivo de tejidos. Asimismo se explica de manera breve una nueva técnica rápida para el diagnóstico de CMV