Use of reverse transcription polymerase chain reaction with colorimetric plate hybridization to detect a cytomegalovirus late spliced mRNA in polymorphonuclear leukocytes from renal transplant patients.

Human cytomegalovirus replication was evaluated in polymorphonuclear leukocytes from ten renal transplant recipients. Three new reverse transcription polymerase chain reactions with plate hybridization suitable for automation were developed for the detection of immediate-early spliced UL123 mRNA, early-late pp65 mRNA, and late spliced UL22 mRNA. The presence of UL22mRNA was found to be significantly associated with the occurrence of cytomegalovirus (CMV) disease.

Comparison of seven RNA extraction methods on stool and shellfish samples prior to hepatitis A virus amplification.

When choosing an extraction method, two parameters have to be considered: recovery of the viral material and elimination or inactivation of inhibitory substances. Seven techniques for extracting hepatitis A virus (HAV) from stool and shellfish samples were compared, in order to identify the protocol most suited to both types of sample and with the best extraction yield. The protocols tested were either techniques for the recovery and purification of total RNA, such as RNAzol, PEG-CETAB, GTC-silica and Chelex, or techniques for isolating specifically HAV using a nucleotide probe or a monoclonal antibody.

Quantification of hepatitis A virus in shellfish by competitive reverse transcription-PCR with coextraction of standard RNA.

To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay.

Simplified reverse transcription polymerase chain reaction procedure with detection by microplate hybridization for routine screening of hepatitis A virus.

Reverse transcription polymerase chain reaction, using either nested or seminested primers, is used extensively for the detection of viruses in small quantities. However, existing methods are prone to false positive reactions. We report here an improved polymerase chain reaction technique based on the use of longer primers (39 nucleotides) with single-step amplification, applied to the detection of hepatitis A in low quantities.

Usefulness of DNA viral load quantification for cytomegalovirus disease monitoring in renal and pancreas/renal transplant recipients.

Background: The purpose of this prospective study was to evaluate the usefulness of quantifying DNA-cytomegalovirus (CMV) load for the diagnosis and monitoring of CMV disease among renal and pancreas transplant patients under immunosuppressive drugs.

Methods: A longitudinal study was conducted among 34 consecutive, unselected renal and pancreas/renal transplanted patients in our unit. During the first 3 posttransplant months, weekly monitoring of CMV infection and CMV disease was done, involving the determination of viremia by the shell vial assay, qualitative DNAemia by semi-nested polymerase chain reaction (PCR) and quantitative DNAemia by the hybrid capture system (HCS), a new and original hybridization method (337 samples were collected for each test).