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. For stool samples, RNAzol, PEG-CETAB, and magnetic beads with antibody allowed detection of the virus in 11/12 and 12/12 of samples. For shellfish samples, three protocols allowed RNA to be extracted in 90% of cases, RNAzol, PEG-CETAB, and GTC-silica. Their rapidity and low cost make RNAzol and GTC-silica the most suitable for routine diagnostic testing. reserved.

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. The quantifiable range with this reproducible method was 10(4) to 10(7) copies of HAV/gram or 400 to 10(6) 50% tissue culture infective doses/ml.

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. While the sensitivity of this technique (10 x the 50% tissue culture infective dose) is equivalent to that of existing methods, it is a simpler procedure, less time consuming, and less susceptible to contamination and therefore provides a more reliable tool for routine diagnosis. Finally, the development of a DNA enzyme immunoassay detection technique and the complete automation of the procedure allow a large number of samples to be processed in clinical laboratories.

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). Qualitative and quantitative DNAemia results were blinded to physicians and three grades of disease were defined according to CMV related symptom occurrence. RESULTS: PCR was the most sensitive (100%) but the least specific (78%) method for the diagnosis of CMV disease. HCS was specific for CMV genome detection, sensitive and reproducible. Blood DNA levels above 60 pg/ml were predictive of severe or moderate CMV disease (sensitivity, 92%; specificity, 100%). A significant decrease in viral load was observed after ganciclovir administration, and a positive PCR or HCS result at the end of the antiviral treatment was associated with relapse of CMV infection or disease. CONCLUSIONS: It is concluded that quantitative DNAemia detection, with this new commercially available method, can predict disease and may be useful for a rational evaluation of ganciclovir preemptive therapy in such patients.

Successful quantification of cytomegalovirus DNA by competitive PCR and detection with capillary electrophoresis.

Human cytomegalovirus (HCMV) is responsible for severe infections in immunocompromised patients. Viral load has recently been identified as one of the major risk factors for subsequent development of HCMV disease. In this context, we developed a protocol allowing rapid, sensitive and precise quantification of HCMV DNA using competitive PCR run to saturation. Long primers were used for amplification, and internal DNA standard was constructed by PCR, with a primer inducing formation of a loop on the target sequence. The obtained fragment differed from the wild one (142 bp) by 6 bp. Quantitative analysis of PCR-amplified HCMV DNA was carried out using an original system combining capillary gel electrophoresis and u.v. detection. This procedure was evaluated on renal transplant recipients, and the results of quantitative PCR were compared with those of viraemia, qualitative DNAemia and HCMV-related symptoms. High levels of HCMV DNA were associated with HCMV-related symptoms, and in all cases a significant decrease of viral load was observed following DHPG treatment. Competitive PCR with capillary electrophoresis detection appears to provide a sensitive quantification method for HCMV DNA in leukocytes and is easily adaptable to routine laboratory use.

Colorimetric microtiter plate hybridization assay using monoclonal antibody for detection of an amplified human immunodeficiency virus target.

Since detection of amplified polymerase chain reaction products is a complicated procedure, a colorimetric microtiter plate hybridization assay was developed to automate specific detection of amplified HIV targets. In this assay, hybridization is seen by an antibody reacting selectively with double-stranded DNA. One hundred and ten amplified products detected by a DNA enzyme immunoassay (DEIA) and by classical hybridization with a digoxigenin-labeling probe, detection sensitivities were less than 10 HIV targets per 10(6) cells. This study demonstrates the specificity and sensitivity of DEIA for detecting amplified HIV targets. The use of the same equipment as for ELISA and the complete automation of the procedure allow a large number of samples to be processed in the clinical laboratory.

Development of RT-semi-nested PCR for detection of hepatitis A virus in stool in epidemic conditions.

The purpose of this study was to determine the efficiency of semi-nested PCR in detecting hepatitis A virus (HAV) RNA. During a 2-year period (1990-1991), HAV RNA was searched for in shellfish from the French Brittany coasts using cRNA and vRNA probes. In January 1992, at the time of a hepatitis A outbreak, 28 stool samples were collected from infected patients (18 adults, 10 children) with anti-HAV IgM. Four samples from subjects with negative HAV serology were used as negative controls. Nucleic acid amplification (reverse-transcription-semi-nested PCR) was performed to detect HAV in stool. HAV RNA was purified by phenol-chloroform extraction and converted to cDNA using reverse transcriptase (Mu-MLV). After amplification, PCR products were visualized on an ethidium-bromide-stained gel and confirmed by hybridization with a specific digoxigenin-labelled oligoprobe. Samples were also studied by molecular hybridization with cRNA and vRNA probes. After onset of the illness, HAV RNA was detected over a longer time period by semi-nested PCR (16/28) than by hybridization (0/28). Even though biological diagnosis of hepatitis A will continue to rely on the detection of anti-HAV IgM, PCR should be useful in certain clinical cases (diagnosis of relapse) and for epidemiological and environmental monitoring of viruses.