PCR-based Method For Detection Research Articles

Reliability of sample collection and laboratory testing for HPV Detection in Men

Analysis of male HPV infection is hindered frequently by the lack of consistency in collection methods and sample adequacy for detection of HPV with molecular methods. Presented here are the reliability of sample collection of male anogenital skin exfoliated cells, as well as reliability of PCR-based HPV detection method and genotyping analysis. Concordance of HPV test for paired collected samples from different anatomical sites was determined. The highest agreement was observed for penile shaft with a kappa ( κ) = 0.75 (95% CI: 0.63–0.86), followed by perianal area ( κ = 0.68, 95% CI: 0.51–0.86); and lowest at the anal canal ( κ = 0.55, 95% CI: 0.35–0.74) and scrotum ( κ = 0.54, 95% CI: 0.40–0.69). The reliability of laboratory testing was highest for detection of oncogenic types ( κ = 0.86, 95% CI: 0.71–1.00) and for multiple-type HPV infections ( κ = 0.84, 95% CI: 0.72–0.95) compared to detection of non-oncogenic HPV types ( κ = 0.64, 95% CI: 0.47–0.82) or single HPV-type detection ( κ = 0.52, 95% CI: 0.32–0.72). In conclusion, the swab method used to obtain skin exfoliated cells is adequate for sample collection, and the specimens can be used reliably for molecular HPV testing.

PCR-based detection of Pol III-transcribed transposons and its application to the rodent model of ultraviolet response

Cellular levels of RNAs containing transposable elements increase in response to various stresses. Polymerase III (Pol III)-dependent transcripts of transposons are different from transposon-containing RNAs generated by read-through Pol II-dependent transcription. Until now, Pol III transcripts were detected by primer extension followed by time-consuming gel electrophoresis. In this paper, we describe a more sensitive PCR-based method for the selective detection of Pol III-transcribed RNAs. The method is based on the difference in sequences at the 5' ends of the Pol II- and Pol III-dependent transcripts. We employed this method to quantify Pol III transcripts of transposon B1 in rodent cells and revealed that their levels are affected by UV irradiation. We therefore conclude that the abundance of the Pol III-transcribed fraction of cellular RNA may serve as marker of stress response and can be conveniently quantified by the method described.

Quantification of circulating endothelial and progenitor cells: comparison of quantitative PCR and four-channel flow cytometry

BackgroundCirculating endothelial cells (CEC) and endothelial precursor cells (CEP) have been suggested as markers for angiogenesis in cancer. However, CEC/CEP represent a tiny and heterogeneous cell population, rendering a standardized monitoring in peripheral blood difficult. Thus, we investigated whether a PCR-based detection method of CEC/CEP might overcome the limitations of rare-event flow cytometry.FindingsTo test the sensitivity of both assays endothelial colony forming cell clones (ECFC) and cord blood derived CD45- CD34+ progenitor cells were spiked into peripheral blood mononuclear cells (PBMNC) of healthy volunteers. Samples were analyzed for the expression of CD45, CD31, CD34, KDR or CD133 by 4-color flow cytometry and for the expression of CD34, CD133, KDR and CD144 by qPCR. Applying flow cytometry, spiked ECFC and progenitor cells were detectable at frequencies ≥ 0.01%, whereas by qPCR a detection limit of 0.001% was achievable. Furthermore, PBMNC from healthy controls (n = 30), patients with locally advanced rectal cancer (n = 20) and metastatic non-small cell lung cancer (NSCLC, n = 25) were analyzed. No increase of CEC/CEP was detectable by flow cytometry. Furthermore, only CD34 and KDR gene expression was significantly elevated in patients with metastatic NSCLC. However, both markers are not specific for endothelial cells.ConclusionQPCR is more sensitive, but less specific than 4-channel flow cytometry for the detection of CEC/CEP cell types. However, both methods failed to reliably detect an increase of CEC/CEP in tumor patients. Thus, more specific CEC/CEP markers are needed to validate and improve the detection of these rare cell types by PCR-based assays.

Multiplex Detection of Human Herpesviruses from Archival Specimens by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

The human herpesviruses are involved in a variety of diseases. Large-scale evaluation of the clinical and epidemiological importance of different herpesviruses requires high-throughput methods. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a method that has a multiplex capacity enabling simultaneous detection of several viruses in a single sample. PCR-based methods for the multiplex detection of all known human herpesviruses were developed on the MALDI-TOF MS system. A variety of 882 archival samples, including bronchoalveolar lavage, conjunctival fluid, sore secretion, blister material, plasma, serum, and urine, analyzed for herpesviruses using PCR-based reference methods, were used to evaluate the MALDI-TOF MS method. The overall concordance rate between the MALDI-TOF MS method and the reference methods was 95.6% (kappa = 0.90). In summary, the MALDI-TOF MS method is well suited for large-scale detection of all known human herpesviruses in a wide variety of archival biological specimens.

Evaluation of RT-PCR and reverse line blot hybridization for detection and genotyping F+ RNA coliphages from estuarine waters and molluscan shellfish

To evaluate a PCR-based detection and typing method for faecal indicator viruses (F+ RNA coliphages) in water and shellfish, and apply the method for better understanding of the ecology and microbial source tracking potential of these viruses. Water and shellfish samples were collected over 3 years at nine estuaries in the East, West and Gulf Coasts of the USA, providing 1033 F+ RNA coliphage isolates. F+ RNA coliphage genotyping rates by reverse transcriptase-PCR-reverse line blot (RLB) hybridization ranged from 94.7% to 100% among estuaries, and were not significantly different in oysters, clams, mussels or water (P = 0.8427). Twenty samples negative by RLB were nucleotide sequenced for confirmation, and to refine RLB probes. More F+ RNA coliphages were genotyped from colder water than warmer waters, while the water salinity did not affect F+ RNA coliphage levels. RT-PCR-RLB was a robust method for detecting and genotyping F+ RNA coliphages from diverse coastal areas, which provided new information on the ecology of F+ RNA coliphages. This performance-validated F+ RNA coliphage method can be used for faecal indicator monitoring and microbial source tracking, to protect recreational bathers and shellfish consumers from exposure to pathogenic virus and their disease risks.

Quantification of beef and pork fraction in sausages by real-time PCR analysis: results of an interlaboratory trial

The performance of quantitative PCR-methods for the determination of beef and pork fraction in sausage was tested in an interlaboratory trial. Twelve different laboratories analysed four sausages of different composition of beef and pork by using four sausages of known fraction of meat as calibrators. Although different PCR-methods were applied, the precision of all results was better than 16% and the trueness better than 25%. The main reason for the good performance is the use of common calibrators emphasizing the importance of the quality of calibrators for molecular analysis. Thus, we conclude, that PCR-based detection methods are suitable for quantitative control of meat fractions in sausages.

Validation of a new seminested PCR-based detection method for Legionella pneumophila

We report a new seminested PCR method for detection of Legionella pneumophila based on the simultaneous amplification of a 387 bp fragment of the dotA gene and a 827 bp recombinant fragment that serves as an internal positive control. This new detection system was validated and its specificity and detection limit were determined. Parallel analysis of 90 environmental samples to compare this method, a real-time PCR method and the standard culture isolation method, demonstrated that this seminested method is useful for the study of L. pneumophila.

Optimization of a 12-Hour TaqMan PCR-Based Method for Detection of Salmonella Bacteria in Meat

We developed a 12-h Salmonella detection method, based on 8 h of preenrichment, followed by automated DNA extraction and a sensitive real-time PCR. The method was optimized to obtain the highest possible yield of cells and DNA. The growth of different Salmonella strains in various preenrichment media and the effects of adding growth-promoting and selective reagents were explored, taking into account their PCR compatibility. The effects of (i) analyzing larger volumes (1 to 5 ml) from preenriched samples and introducing wash steps prior to DNA extraction, (ii) regulating the amount of paramagnetic particles (increasing it from 60 to 90 microl) in the DNA extraction, (iii) eluting the DNA in reduced volumes (25 or 50 microl rather than 100 microl), and (iv) increasing the PCR template volume (from 5 to 20 microl) were investigated. After 8 h of preenrichment, buffered peptone water yielded the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 microl were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 microl) had no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 mul improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples, with a relative accuracy of 99%, a relative sensitivity of 98%, and a relative specificity of 100%.

Amplification of Three Unlinked Regions of Honey Bee (Hymenoptera: Apidae) DNA with Polymorphisms at High Frequencies in East European Subspecies

Abstract Honey bee, Apis mellifera L. (Hymenoptera: Apidae) DNA has alleles, at three polymorphic restriction sites, which were initially found almost exclusively in samples from eastern Europe. These markers were first detected with three labeled, cloned, probes on DNA blots. In this study, the polymorphic sites were made easier to analyze with the polymerase chain reaction (PCR). The amplification primers and conditions are described. The regions with the polymorphic sites were mapped within the sequences of the honey bee genome and were found within three separate linkage groups. The genomic locations of the defining restriction sites and PCR primers are given. With the PCR-based method of detection, additional samples of bees from Europe and Africa were examined. Within the eastern European group, the three markers were at high frequencies in Italian bees (subspecies Apis mellifera ligustica Spinola), two markers were at high frequencies and one marker was at a lower frequency in Carniolan bees (Apis ...

Improved One-step Colony PCR Detection of Vibrio harveyi

Vibrio harveyi strains are pathogenic to a wide range of marine fish and shellfish, having a significant negative economic impact on aquaculture worldwide. However, a reliable and rapid method of detecting V. harveyi has yet to be established. We aimed to construct an improved one-step detection method for V. harveyi. Reanalysis of 16S rRNA gene sequences of type strains of V. harveyi revealed a unique consensus region compared to related Vibrio species. Using a VHARF-VHARR primer set from this region, a V. harveyi-specific PCR-based detection method was established and could differentiate V. harveyi from related species such as V. campbellii, V. rotiferianus, and V. alginolyticus. Furthermore, the new method (optimal amplification with 20 core cycles of (94°C-30 s, 60°C-30 s, 72°C-30 s)) could be applied to the identification of V. harveyi strains of colonies. This PCR was able to detect V. harveyi grown on plates in the environment within 3 days without bacterial isolation, DNA extraction, or the assistance of biochemical tests. The specificity and rapidity of the detection is reliable enough to understand the ecology of V. harveyi in environments, and to predict outbreaks of mass mortality caused by V. harveyi in aquaculture.

A gene expression profile of tumor suppressor genes commonly methylated in bladder cancer

The functional relationship between promoter hypermethylation and gene inactivation has been demonstrated for few genes only. We examined the promoter methylation status of two important tumor suppressor genes APAF-1 and DAPK-1 in bladder cancer as well as the mRNA expression pattern of these two genes for possible correlation between promoter hypermethylation and transcriptional repression. The methylation status and mRNA expression levels were related to clinicopathological features in 34 patients with transitional cell carcinoma (TCC) of the bladder with a median clinical follow-up of more than 45 months. Tissue from ten patients with nonmalignant disease served as a control group. Quantitative real-time PCR-based detection methods were used for determination of the normalized index of methylation (NIM) as well as the mRNA expression level. APAF-1 and DAPK-1 methylation and mRNA expression was observed in all tumor and normal control samples investigated. Methylation (NIM) levels were significantly higher in tumor tissue for APAF-1 and DAPK-1, but median mRNA expression levels did not differ significantly comparing tumorous and non tumorous tissue. No correlation between expression levels of APAF-1 and DAPK-1 mRNA and tumor stage or grade was observed. However, in superficial TCC a strong correlation between higher NIM levels and lower mRNA expression of the APAF-1 gene was observed (P = 0.014). Our results, although preliminary, provide first time in vivo expression analysis of the APAF-1 gene in bladder cancer specimen, suggesting expression control by promoter methylation in early stage tumor disease of the bladder.

A PCR-based method for detection and quantification of small RNAs

Recent cloning efforts have identified hundreds of thousands of small RNAs including micro RNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs). These non-coding small RNAs need to be further validated and characterized by detecting and quantifying their expression in different tissues and during different developmental courses. A simple, accurate, and sensitive method for small RNA expression profiling is in high demand. Here, we report such a PCR-based method.

Large subunit ribosomal RNA gene variation and sequence heterogeneity of Dinophysis (Dinophyceae) species from Scottish coastal waters

The purpose of the study was to investigate the genetic diversity of Dinophysis species from around the Scottish coast, with a view to an improved understanding of the dynamics and identification of this genus in Scottish waters. Single-cell PCR amplification with direct sequencing was performed on a total of 441 Dinophysis cells isolated from both live and Lugol's fixed plankton net samples. Universal eukaryotic primers were used to amplify the large subunit (LSU) ribosomal RNA (rRNA) gene of the Dinophysis isolates, with a frequency of PCR success of 26% for non-fixed and 48% for fixed samples. From this a total of 30 isolates were selected for this study and the D1–D2 region of the LSU-rRNA gene sequenced for phylogenetic analysis. No significant correlation could be made between geographical location and LSU sequence, although some regional sequence heterogeneity was observed within the Dinophysis acuta species. LSU sequence data was used to design Dinophysis genus specific and Dinophysis clade-specific primers primarily to ensure clean sequences from universal D1–D2 amplicons without a requirement for cloning. Three clade-specific primers designed to a region within the D2 hypervariable region of the LSU-rRNA gene allowed discrimination of Dinophysis acuminata/ norvegica from Dinophysis tripos/caudata and Dinophysis fortii/ acuta. In two isolates, SC359 ( D. tripos) and LC58 ( D. acuta), nested PCR products were observed with both the expected clade-specific primer, and Dasd-R2, the D. acuminata/ norvegica clade-specific primer. Cloning and sequence analysis suggested that these amplicons were genuine “ D. acuminata-like” sequences and their presence, albeit at a low frequency within different Dinophysis species, indicated that individual Dinophysis cells possess heterologous copies of the LSU-rRNA gene that are similar to LSU sequences normally associated with D. acuminata. The nature of the process that generated these hybrid cells, the frequency of such events and their importance is as yet unknown, but may provide a cautionary note for the development of PCR-based species specific detection methods.

Detection of genetically modified rice: a construct-specific real-time PCR method based on DNA sequences from transgenic Bt rice

Genetically modified rice varieties developed in China are close to approval for agricultural cultivation and production. However, so far no method has been reported for specific detection of transgenic varieties of this crop. In the present study, rice seeds assumed to consist of field-tested Bt rice (‘Anti-pest Shanyou 63’ and ‘Anti-pest Jinyou 63’) were used as reference material to determine transgenic DNA sequences. The transition between the cryIA(b) and cryIA(c) fusion gene and the nopaline synthase terminator (nos) sequence was used to develop a construct-specific real-time PCR based detection method. This Bt rice specific detection system was combined with a recently published quantitative real-time PCR method for the rice-specific (Oryza sativa L.) reference gene gos9. The complete PCR assay for detection of transgenic Bt rice was in-house validated and the limit of quantification was found to be below 0.1% Bt rice relative to the rice content. Application of the PCR assay should allow more precise detection of transgenic rice varieties in imported food products which are so far not approved in the EU.

A nested PCR-based method for detection ofStreptococcus agalactiae16S rRNA in milk and its application

AbstractBovine mastitis caused byStreptococcus agalactiaeis mainly subclinical and therefore can be diagnosed only in the laboratory. A nested polymerase chain reaction (PCR)-based method for specific, sensitive and rapid detection ofS. agalactiaein raw milk was developed. The general streptococci primers, which anneal to conserved areas within the 16S rRNA subunit gene, were used as positive controls. The specificity ofS. agalactiaeprimers is based on various areas within conserved areas of the 16S rRNA genes ofS. agalactiae. Results have indicated that the method enables the detection of 1 CFU/ml ofS. agalactiaein raw milk after enrichment, followed by DNA extraction using a rapid and simple procedure developed for this purpose, and specific PCR reaction. The method developed can be used efficiently in the early infectious status investigation ofS. agalactiaein the dairy herd and in prevention and control ofS. agalactiaespread in a herd.

Greyhound meningoencephalitis: PCR-based detection methods highlight an absence of the most likely primary inducing agents

Greyhound meningoencephalitis is currently classified as a breed-associated idiopathic central nervous system inflammatory disorder. The non-suppurative inflammatory response can be distinguished from the other breed-associated disorders based on histopathology and lesion topography, however the nature of the response primarily suggests a viral infection. In the present study PCR and RT-PCR technologies were employed on frozen cerebral tissue from confirmed cases of meningoencephalitis to target specific viruses and protozoa likely to be implicated and to exclude the presence of bacterial 16SrRNA. Secondly, degenerate primers were used to detect viruses of the herpesvirus and flavivirus families. In addition cerebral tissues were probed for West Nile Virus. Viral nucleic acid sequences to Borna disease virus, to louping ill, tick borne encephalitis, West Nile and other flaviviruses were not detected. Canine distemper virus was detected in one animal with 97% homology to strain A75/15. Degenerate PCR for herpesviruses detected viral amplification products in one animal with 90% homology to canine herpesvirus DNA polymerase gene. Protozoal amplification products were only detected in a single dog with pathological confirmation of a combination of lesions of greyhound meningoencephalitis and a protozoal encephalomyelitis. Neospora was confirmed with sequence homology to Austrian strain 1. Bacterial 16SrRNA was not detected. The present study supports previous observations that many of the known microbial causes of canine meningoencephalitis are not involved. Findings could reflect that the causal agent was not specifically targeted for detection, or that the agent is at undetectable levels or has been eliminated from brain tissue. The potential roles of genetics and of molecular mimicry also cannot be discounted.

Rapid PCR-based method for detection and differentiation of Didymiaceae and Physaraceae (myxomycetes) in environmental samples

Ecological studies of myxomycetes have been limited by the absence of universal cultivation techniques and the lack of life stage independent identification methods. We designed a novel PCR primer pair for the specific amplification of small subunit ribosomal RNA gene of Didymiaceae and Physaraceae. The primers produced amplicons from 192 fruiting body samples belonging to 10 genera. Twenty-four samples yielded longer fragments and sequence analysis revealed the presence of intron(s). As for the exonic regions, while sequence heterogeneities within a single species/varietas/forma were frequently observed, identical sequences were obtained only from identical species/varietas. The effectiveness of this primer pair in the analysis of morphologically unidentifiable samples was confirmed with the applications to samples of environmental plasmodium/sclerotium and soil. Denaturing gradient gel electrophoresis analysis was also tested with the soil samples. The results presented here demonstrate this PCR-based method can facilitate further ecological studies of Physaraceae and Didymiaceae in the environment.

Staphylococcal Enterotoxin D Production by Staphylococcus aureus FRI 100

Staphylococcus aureus FRI 100 is commonly used as a control strain for staphylococcal enterotoxin A (SEA) assays. When FRI 100 was used in PCR-based enterotoxin detection methods, the strain gave a positive result for both SEA and staphylococcal enterotoxin D (SED). Production of SED was confirmed by testing concentrated and unconcentrated culture supernatants with the TECRA staphylococcal enterotoxin visual immunoassay. SED was detected after 24 h of growth in Trypticase soy broth. Primers were created to amplify the entire sed gene by PCR for subsequent sequencing. The sequenced gene showed high similarity to a previously sequenced sed gene. The SED-like gene in FRI 100 exhibited four point mutations and two deletions. Changes in the FRI 100 open reading frame altered the primary structure of the SED-like protein, allowing for coding of only the first 150 amino acids followed by a stop codon. Because the SED active site is at the proximal end, where there was no change in DNA sequence, we conclude FRI 100 produces a variant form of SED. It is necessary to note that, when using FRI 100 as an SEA control strain, it does produce a variant of the SED protein, which exhibits immunological activity, and the sed-like gene is detected by commonly used PCR primers. This phenomenon may be an important general consideration when using PCR to characterize strains of toxin-producing S. aureus. S. aureus enterotoxin–positive PCR results should be confirmed by immunological techniques.

Detecting key parasitoids of lepidopteran pests by multiplex PCR

The use of diagnostic polymerase chain reaction (PCR) is a valuable approach to study host–parasitoid interactions. It circumvents problems inherent to rearing parasitoids from Weld-collected hosts or host dissection. Here, we present a PCR-based detection method for Diadegma semiclausum, Cotesia glomerata, and Cotesia rubecula, which are key parasitoids of the three lepidopteran cabbage pests Plutella xylostella, Pieris brassicae, and Pieris rapae, respectively. Primer pairs for the three parasitoid species and Pl. xylostella were developed: they were used either separately in singleplex PCR or combined in multiplex PCR to (1) screen simultaneously for the two Cotesia species or to (2) detect the parasitoid D. semiclausum and identify its host Pl. xylostella in one PCR. The new idea to simultaneously identify parasitoid and host by molecular markers is useful when the host, in our case early-instar larvae of Pl. xylostella, is morphologically diYcult to distinguish from other host species also occurring in the same habitat. Concentration–response trials revealed comparable detection sensitivity of singleplex and multiplex PCR, with detection limits ranging from 0.03 to 2.2 pg of parasitoid DNA/l PCR. Furthermore, the different developmental time of immature D. semiclausum and C. glomerata did not inXuence parasitoid detection success in either assay type. Based on multiplex PCR screening of Weld-collected caterpillars, we found in Pl. xylostella, P. brassicae, and P. rapae parasitism rates of 33.4% by D. semiclausum, 52% by C. glomerata, and 53.4% by C. rubecula, respectively. © 2006 Elsevier Inc. All rights reserved.

New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis

BackgroundJohne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples.ResultsOur confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system.ConclusionWe recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used.