Semi-nested PCR Assay Research Articles

Evaluation of the AmpliSensor PCR and the SHARP signal detection system for the early prediction of symptomatic CMV infection in solid transplant recipients.

Cytomegalovirus (CMV) is associated with high morbidity and mortality in transplant patients. Specific antiviral treatment at an early stage of CMV infection may effectively ameliorate, but not eliminate CMV disease in these patients. Presently, the pp65 antigenemia test on peripheral leukocytes is the method most widely used for predicting and monitoring transplant patients for active CMV infection. Nucleic acid amplification methods are less well defined since they lack standardisation. A seminested fluorometric PCR assay (AmpliSensor-CMV, BAG, Germany) and a one-step PCR with a signal-amplification step (SHARP, Abbott, Germany) specific for the fragments of the CMV UL 122 and UL 123 genes, respectively, were evaluated for the early diagnosis of CMV infection. A total of 26 recipients of heterogeneous solid organs were monitored prospectively for a median of 99 days after transplantation. By testing 371 clinical samples parallel with the pp65-antigen assay and IgM and IgG EIA assays the sensitivity, specificity, correlation and quantitation potential of both PCRs was evaluated. Eight out of 26 patients developed active CMV infection. A total of 48 samples of these patients exceeded a CMV-DNA load threshold of 15 genome equivalents/10(5) leukocytes (AmpliSensor-CMV) and 41 samples exceeded the critical cut-off for the SHARP system. The AmpliSensor PCR exceeded its threshold consistently before the clinical onset of CMV disease (median 8 days). There was very good agreement between symptomatic CMV infection in patients and AmpliSensor-PCR, SHARP PCR, and pp65-antigen results (kappa-coefficient > 0.900). IgM and IgG EIA showed moderate agreement (kappa-coefficient = 0.591 and 0.552, respectively). Both PCRs and pp65 antigen assay correlated significantly better with CMV disease than serodiagnosis. The AmpliSensor PCR allowed more precisely than the SHARP system a quantitative determination of viral load and an early and reliable prediction of active CMV infection. The use of AmpliSensor PCR may improve the diagnosis and management of active CMV infection in organ transplant recipients.

Detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples by a seminested PCR assay.

A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA and flaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8, 700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as </=3 CFU per g of food could be detected with samples subjected to overnight enrichment, while variable results were obtained for samples analyzed without prior enrichment. This rapid and sensitive nested PCR assay provides a useful tool for specific detection of C. jejuni or C. coli in drinking water, as well as environmental water, sewage, and food samples containing high levels of background organisms.

Application of pbp1A PCR in identification of penicillin-resistant Streptococcus pneumoniae.

A seminested PCR assay, based on the amplification of the pneumococcal pbp1A gene, was developed for the detection of penicillin resistance in clinical isolates of Streptococcus pneumoniae. The assay was able to differentiate between intermediate (MICs = 0.25 to 0.5 microgram/ml) and higher-level (MICs = >/=1 microgram/ml) resistance. Two species-specific primers, 1A-1 and 1A-2, which amplified a 1,043-bp region of the pbp1A penicillin-binding region, were used for pneumococcal detection. Two resistance primers, 1A-R1 and 1A-R2, were designed to bind to altered areas of the pbp1A gene which, together with the downstream primer 1A-2, amplify DNA from isolates with penicillin MICs of >/=0.25 and >/=1 microgram/ml, respectively. A total of 183 clinical isolates were tested with the pbp1A assay. For 98.3% (180 of 183) of these isolates, the PCR results obtained were in agreement with the MIC data. The positive and negative predictive values of the assay were 100 and 91%, respectively, for detecting strains for which the MICs were >/=0.25 microgram/ml and were both 100% for strains for which the MICs were >/=1 microgram/ml.

Rapid feline immunodeficiency virus provirus quantitation by polymerase chain reaction using the TaqMan® fluorogenic real-time detection system

An improved quantitative polymerase chain reaction (qPCR) method based on a combination of real-time detection and the 5′–3′ nuclease activity of the Taq DNA polymerase was developed to quantify the provirus load of feline immunodeficiency virus (FIV), a lentivirus of veterinary importance and an animal model for AIDS research. Two fluorogenic probes were designed to detect FIV provirus in genomic DNA of peripheral lymphocytes and tissues infected with different FIV subtypes. The most sensitive assay can detect one copy of FIV provirus. The assay showed excellent precision within-run and between-runs. Comparison of the TaqMan system with a conventional seminested PCR assay revealed a comparable detection limit and good correlation. Furthermore the design of the two probes allowed the detection of various FIV isolates of clade A and B.

Gene conversion can create new MHC alleles.

It is possible to measure gene conversion of MHC genes with the help of a semi-nested PCR assay. Several considerations are of utmost importance when such an assay is set up. Using this assay, we have found that gene conversion occurs in MHC class II genes in mouse sperm, but not in somatic cells tested. Although this gene conversion occurs in germline cells, it is already completed in spermatogonia, and consequently is mitotic event unlinked to meiosis. The frequency of gene conversion events in MHC class II genes varies strongly from one allele to another, with the highest detected frequencies as high as 1/40,000 for an individual heterozygous for both donor and acceptor sequences. Deletions or insertions in one gene relative to the other seem to lower the efficiency of gene conversion considerably. Stretches within MHC genes amenable to gene conversion are located in CpG clusters, whereas MHC genes not involved in gene conversion have background CpG levels. DNA damage, either chemical or radiation induced, increases the frequency of gene conversion of MHC class II genes in cultured cells of the fibroblastoid lineage. The effect of chemical DNA damage seems roughly dose dependent, whereas irradiation has a maximal effect at low doses.

Detection of Helicobacter pylori in stool specimens by PCR and antigen enzyme immunoassay.

A highly sensitive seminested PCR assay to detect Helicobacter pylori DNA in feces was developed. PCR with stool specimens and a novel antigen enzyme immunoassay (EIA) for H. pylori detection in feces were evaluated as diagnostic tools and in follow-up with samples from 63 infected and 37 noninfected persons. Infected individuals received eradication therapy followed by endoscopic follow-up 35 days after the start of treatment. At that time, a second stool specimen was obtained from 55 of these patients. Before eradication, the sensitivity of PCR was 93.7% and that of EIA 88.9%. Specificities were 100 and 94.6%, respectively. Of the 55 follow-up specimens, 41 originated from patients from whom H. pylori had been eradicated. Of these, 21 were still positive by PCR and 13 were positive by EIA, indicating that 1 month may be too short a period for follow-up evaluation of stool specimens by these tests.

A highly sensitive seminested-PCR assay for diagnosis of bancroftian filariosis

A highly sensitive seminested-PCR assay for diagnosis of bancroftian filariosis

Rapid detection of penicillin-resistant Streptococcus pneumoniae in cerebrospinal fluid by a seminested-PCR strategy.

A seminested-PCR assay, based on the amplification of the pneumococcal penicillin-binding protein 2B gene (pbp2B), was developed for the detection of penicillin-resistant and -susceptible pneumococci in cerebrospinal fluid (CSF) specimens. Species-specific primers (P5 and P6) which amplified a 682-bp conserved region of the transpeptidase-encoding region of the pbp2B gene were used. Four "resistance" primers were designed to bind to altered areas of the pbp2B gene identified in penicillin-resistant South African wild-type strains. Together with the downstream primer P6, the upstream resistance primers amplified fragments which were used to detect the presence of penicillin resistance. This system identified all 35 of the S. pneumoniae isolates evaluated, including strains of 11 different serotypes and a range of penicillin-resistant and -susceptible strains. The specificity of the assay was demonstrated by its inability to amplify DNA from other bacterial species which commonly cause meningitis. It was possible to detect pneumococcal DNA from culture-negative CSF inoculated with 2.5 pg of purified DNA or 18 CFU. Analysis of 285 CSF specimens showed that PCR detected the pneumococcus in 18 samples positive by culture, including the identification of four penicillin-resistant isolates. The positive predictive value and the negative predictive value of the assay were each 100%.

Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water.

The presence of PCR inhibitors in water samples is well known and contributes to the fact that a practical PCR assay has not been developed for legionella surveillance. In this study, we devised a new seminested PCR assay for detection of Legionella spp. in water samples as a means of overriding the PCR inhibitors without loss of sensitivity. The seminested PCR assay utilized primers to amplify the 16S rRNA gene (LEG primers) of 39 Legionella spp. The assay was specific to legionellae, and the sensitivity was 1 fg of extracted Legionella DNA in laboratory examination. To evaluate the feasibility and sensitivity of the PCR assay in identifying the presence of legionellae, it was used to survey Legionella contamination in the water of 49 cooling towers of 32 hospitals. A commercially available EnviroAmp Legionella kit and a culture method were also used in the survey for comparison with the seminested PCR assay. The detection rates of legionellae in the samples were 91.8% (45 of 49) by the PCR assay and 79.5% (39 of 49) by the culture method. The EnviroAmp kit revealed that 30.6% of the water samples (15 of 49) contained inhibitors of the PCR amplification. However, the seminested PCR assay could produce the Legionella-specific DNA bands in 14 of the 15 samples. Although 8 of the 14 samples were positive in the first-step PCR, 6 of the 14 samples became positive in the second-step PCR. These results suggest that the effect of PCR inhibitors in samples, if any, can be reduced because of the dilution of the sample in the second-step PCR and that sensitivity of detection can be increased by the second-step PCR. Thus, the seminested PCR assay with LEG primers to amplify the 16S rRNA gene of 39 Legionella spp. was a practical and sensitive method to detect Legionella spp. in water samples.

Preproinsulin I and II mRNAs and insulin electron microscopic immunoreaction are present within the rat fetal nervous system

Insulin-like substance has been found within the nervous system. In the rat, preproinsulin II mRNA was shown within the brain and preproinsulin mRNA within the retina. The present study demonstrates the presence of preproinsulin mRNA within the 15, 17 and 19 day gestational age fetal rat brain, spinal cord and dorsal root ganglia (DRG), employing RNA template-specific polymerase chain reaction (RS-PCR), semi-nested PCR and RNase protection assay. Preproinsulin I mRNA was present in the 17 and 19 day gestational age brain, spinal cord and DRG, and only in the brain of the 15 day gestational age brain. Preproinsulin II mRNA was present in all the gestational ages studied in the brain, spinal cord and DRG. The RS-PCR and the semi-nested PCR demonstrated products that co-migrated with the pancreatic control. The semi-nested products were characterized as preproinsulin I and II by restriction enzyme digestion and sequence. RNase protection assay using specific cRNA for preproinsulin I and II showed a band that co-migrated with pancreatic preproinsulin I and II mRNAs, and confirmed the PCR results. In addition, insulin receptor mRNA was detected by RS-PCR. Ultrastructural studies showed insulin immunoreaction within the endoplasmic reticulum, Golgi apparatus, cytoplasm, axon, dendrites, and in relation to the synapses. Thus, we demonstrated the presence of preproinsulin I and II mRNA, insulin receptor mRNA and insulin immunoreaction within the rat fetal central and peripheral nervous system.

Preproinsulin I and II mRNAs and insulin electron microscopic immunoreaction are present within the rat fetal nervous system

Insulin-like substance has been found within the nervous system. In the rat, preproinsulin II mRNA was shown within the brain and preproinsulin mRNA within the retina. The present study demonstrates the presence of preproinsulin mRNA within the 15, 17 and 19 day gestational age fetal rat brain, spinal cord and dorsal root ganglia (DRG), employing RNA template-specific polymerase chain reaction (RS-PCR), semi-nested PCR and RNase protection assay. Preproinsulin I mRNA was present in the 17 and 19 day gestational age brain, spinal cord and DRG, and only in the brain of the 15 day gestational age brain. Preproinsulin II mRNA was present in all the gestational ages studied in the brain, spinal cord and DRG. The RS-PCR and the semi-nested PCR demonstrated products that co-migrated with the pancreatic control. The semi-nested products were characterized as preproinsulin I and II by restriction enzyme digestion and sequence. RNase protection assay using specific cRNA for preproinsulin I and II showed a band that co-migrated with pancreatic preproinsulin I and II mRNAs, and confirmed the PCR results. In addition, insulin receptor mRNA was detected by RS-PCR. Ultrastructural studies showed insulin immunoreaction within the endoplasmic reticulum, Golgi apparatus, cytoplasm, axon, dendrites, and in relation to the synapses. Thus, we demonstrated the presence of preproinsulin I and II mRNA, insulin receptor mRNA and insulin immunoreaction within the rat fetal central and peripheral nervous system.

Nested-Polymerase Chain Reaction for the Detection ofHelicobacter pyloriInfection with Novel Primers Designed by Sequence Analysis of Urease A Gene in Clinically Isolated Bacterial Strains

We have established a highly sensitive semi-nested PCR assay for the detection ofH. pyloriinfection using gastric juice samples, which can be aspirated with disposable nasogastric tubes. The primers targetingH. pyloriurease A gene were designed based on the sequence conservation analysis of sixteenH. pyloristrains isolated from Japanese patients. The efficacy of the PCR assay, designated as the URA-PCR, was confirmed byin vitroandin vivoassessments. Its sensitivity was 97.5% in the gastric juice samples aspirated from forty patients with provenH. pyloriinfection, and was significantly higher than that obtained with previously described PCR assays.

Genotyping of rotaviruses isolated from sewage.

Rotaviruses from environmental samples have been genotyped by a seminested reverse transcription PCR assay with serotype-specific primers derived from variable regions of gene 9, which produce different characteristic segment sizes for serotypes 1 to 4. The method enabled the detection and identification of type 1, 2, and 3 group A rotaviruses in sewage.