Direct Polymerase Chain Reaction Detection Research Articles

Comparison of extraction-based and elution-based polymerase chain reaction testing, and automated and rapid antigen testing for the diagnosis of severe acute respiratory syndrome coronavirus 2.

We aimed to compare the differences in testing performance of extraction‐based polymerase chain reaction (PCR) assays, elution‐based direct PCR assay, and rapid antigen detection tests for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). We used nasopharyngeal swab samples of patients with coronavirus disease 2019 (COVID‐19). We used the MagNA Pure 24 System (Roche Diagnostics K.K.) or magLEAD 12gC (Precision System Science Co., Ltd.) for RNA extraction, mixed the concentrates with either the LightMix Modular SARS‐CoV PCR mixture (Roche Diagnostics K.K.) or Takara SARS‐CoV‐2 direct PCR detection kit (Takara Bio Inc.), and amplified it using COBAS® z480 (Roche Diagnostics K.K.). For elution‐based PCR, we directly applied clinical samples to the Takara SARS‐CoV‐2 direct PCR detection kit before the same amplification step. Additionally, we performed Espline SARS‐CoV‐2 (Fuji Rebio Co., Ltd.) for rapid diagnostic test (RDT), and used Lumipulse SARS‐CoV‐2 antigen (Fuji Rebio Co., Ltd.) and Elecsys SARS‐CoV‐2 antigen (Roche Diagnostics K.K.) for automated antigen tests (ATs). Extraction‐based and elution‐based PCR tests detected the virus up to 214–216 and 210 times dilution, respectively. ATs remained positive up to 24–26 times dilution, while RDT became negative after 22 dilutions. For 153 positive samples, positivity rates of the extraction‐based PCR assay were 85.6% to 98.0%, while that of the elution‐based PCR assay was 73.2%. Based on the RNA concentration process, extraction‐based PCR assays were superior to elution‐based direct PCR assays for detecting SARS‐CoV‐2.

Prevalence of Shiga Toxin–Producing Escherichia coli and Associated Virulence Genes in Feces of Commercial Feedlot Cattle

The objective of this study was to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) serogroups and associated virulence genes in feces of commercial feedlot cattle. During March to May 2011, fecal samples were collected from individual cattle (n=960) in 10 cohorts (cattle subpopulations within a feedlot) comprising 17,148 total steers that originated from 48 backgrounding operations in six U.S. states. Fecal samples were enriched in E. coli broth and subjected to two detection protocols: (1) an 11-gene multiplex polymerase chain reaction (PCR) that identifies seven O serogroups (O26, O45, O103, O111, O121, O145, and O157) and four virulence genes (stx1, stx2, eae, and ehxA) applied to extracted total DNA ("direct PCR"); and (2) cultural procedures that involve immunomagnetic separation (IMS) with O26, O103, and O111 beads, plating on a nondifferential MacConkey agar, followed by the multiplex PCR of pooled colonies ("culture-based method"). Generalized linear mixed models were used to adjust prevalence estimates for clustering. Based on direct PCR detection, O157 (49.9%) was the most prevalent O serogroup followed by O26 (20.3%), O103 (11.8%), O121 (10.7%), O45 (10.4%), O145 (2.8%), and O111 (0.8%). Cumulative adjusted prevalence estimates were 22.3, 24.6, and 0.01% for O26, O103, and O111 serogroups, respectively, based on culture-based methods. However, prevalence varied significantly by cohort (p-values<0.05) for O26, O121, and O157 based on direct PCR, and for O26, O103, and O111 serogroups based on culture-based methods. Results of this study indicate that all seven STEC serogroups were identified in feedlot cattle feces, with O157, O26, and O103 being the most prevalent serogroups. A substantial proportion of serogroup-positive samples did not harbor Shiga toxin genes; thus, additional elucidation of the potential human health risk is required. Further evaluation of diagnostic methods for non-O157 STEC is needed given their impact on prevalence estimation.

Polymerase chain reaction assay for the detection of Acinetobacter baumannii in endotracheal aspirates from patients in the intensive care unit

We aim to evaluate the efficacy of polymerase chain reaction (PCR) to detect Acinetobacter baumannii in endotracheal aspirates. Endotracheal aspirates and clinical data were collected from patients who were admitted to the intensive care unit of Taipei Veterans General Hospital between April 1 and August 31 in 2006. Bacterial isolates from endotracheal aspirate cultures were phenotypically identified as Acinetobacter calcoaceticus-A baumannii complex using the API ID 32GN system. The presence of A baumannii in the aspirate was also directly detected by multiplex PCR. Ten of the 114 endotracheal aspirate cultures were positive for A calcoaceticus-A baumannii complex, and only nine of the isolates were confirmed as A baumannii by the multiplex PCR. Direct PCR detection showed that 40 (35.1%) of the endotracheal aspirates were positive for A baumannii. Using positive culture of A baumannii as the gold standard, the sensitivity of direct PCR detection was 100% (6 of 6), the specificity was 70.4% (38 of 54), the positive predictive value was 27.3% (6 of 22), and the negative predictive value (NPV) was 100% (38 of 38) among patients with A baumannii pneumonia. Among patients with A baumannii colonization, the sensitivity of direct PCR detection was 100% (3 of 3), the specificity was 70.6% (36 of 51), the positive predictive value was 16.7% (3 of 18), and the NPV was 100% (36 of 36). Direct PCR detection of A baumannii in endotracheal aspirates has a high sensitivity and NPV as compared with culture-based methods. Further studies are needed to determine the clinical applicability of this rapid detection test.

Rapid sequencing and analysis for pathogen confirmation

Genetic amplification methods and especially those based on the polymerase chain reaction (PCR) are revolutionising diagnostic microbiology. Genetic amplification is rapidly becoming the method of choice to detect viruses, ?single pathogen? infections such as Chlamydia and Gonorrhoea, slow growing or difficult-to-grow bacteria such as mycobacteria and legionella, or toxigenic bacteria such as Shigatoxigenic Escherichia coli and Clostridium difficile. This trend has been accelerated with the use of fluorescent, probe-based, real-time PCR, high-throughput thermocyclers and the use of robotics. Traditional biochemistry-based identification is adequate for many bacterial isolates, as is the specificity afforded by real-time probes for direct PCR detection. However, the sequencing of amplified products (amplicons) is also often performed where increased resolution is required, even though longer amplicons than those generated by real-time PCR are required. Two benefits are clear. Firstly, sequence is constructed solely from the four nucleotides so can be likened to a digital signal. In comparison, biochemical identifications are more like an analogue signal, where weak or equivocal reactions can create ambiguity. Secondly, the resolution afforded by biochemical reactions (less than 100 character states i.e. 48 reactions that can be positive or negative) is easily exceeded by sequencing (2000 character states for a 500 bp sequence i.e. 500 sites which can contain one of four nucleotides). Further, the sequence is reproducible and not dependent on variables such as the bacterial growth conditions and easily lends itself to computer-based analysis.

Direct multiplex reverse transcription-nested PCR detection of influenza viruses without RNA purification.

This paper describes the development a of direct multiplex reverse transcription-nested polymerase chain reaction (PCR) method, devised for simultaneous detection and typing of influenza viruses. This method combines the direct reverse transcription reaction without RNA purification with the enhancement of sensitivity and specificity of nested PCR. The method successfully detected three major human influenza viruses: influenza virus A subtype 1 (H1N1) and subtype 3 (H3N2), and influenza B virus (B). The minimum number of virus particles (pfu/ml) necessary for detection in spiked saliva samples was 200 (H1N1), 140 (H3N2), and 4.5 (B). The method's sensitivity and simplicity will be convenient for use in clinical laboratories for the detection and subtyping of influenza and possibly other RNA viruses.

Direct Detection of Insertion/Deletion Polymorphisms in an Autosomal Region by Analyzing High-Density Markers in Individual Spermatozoa

Direct polymerase chain reaction (PCR) detection of insertion/deletion (indel) polymorphisms requires sample homozygosity. For the indel polymorphisms that have the deletion allele with a relatively low frequency in the autosomal regions, direct PCR detection becomes difficult or impossible. The present study is, to our knowledge, the first designed to directly detect indel polymorphisms in a human autosomal region (i.e., the immunoglobulin V(H) region), through use of single haploid sperm cells as subjects. Unique marker sequences (n=32), spaced at approximately 5-kb intervals, were selected near the 3' end of the V(H) region. A two-round multiplex PCR protocol was used to amplify these sequences from single sperm samples from nine unrelated healthy donors. The parental haplotypes of the donors were determined by examining the presence or absence of these markers. Seven clustered markers in 6 of the 18 haplotypes were missing and likely represented a 35-40-kb indel polymorphism. The genotypes of the donors, with respect to this polymorphism, perfectly matched the expectation under Hardy-Weinberg equilibrium. Three V(H) gene segments, of which two are functional, are affected by this polymorphism. According to these results, >10% of individuals in the human population may not have these gene segments in their genome, and approximately 44% may have only one copy of these gene segments. The biological impact of this polymorphism would be very interesting to study. The approach used in the present study could be applied to understand the physical structure and diversity of all other autosomal regions.

High levels of congenital transmission of Toxoplasma gondii in a commercial sheep flock

Our current understanding of congenital transmission of Toxoplasma gondii from ewe to lamb dictates that infection frequently results in abortion and the death of the developing foetus, that the birth of live infected lambs occurs rarely and that the cat is the predominant source of infection in ewes. Using direct polymerase chain reaction detection of T. gondii, we report high levels of congenital transmission occurring in a commercially managed sheep flock. We sampled foetal-derived placental tissue and tissues from aborted lambs and showed that congenital transmission was detected in these tissues from 61% of all pregnancies. Where pregnancies resulted in the death of one or more lambs, T. gondii was detected in the lamb tissue for all but one of 18 (94%) pregnancies. Of the successful pregnancies resulting in the birth of live lambs we were able to detect T. gondii in foetal-derived placental tissue from 37 of 70 (42%) pregnancies. These results show that congenital transmission is occurring in a high percentage of lambings including normal healthy lambings, at this farm, suggesting that this route of transmission from generation to generation may be much more significant than that reported previously. These results may have implications for sheep husbandry and future epidemiological studies of T. gondii.

Direct polymerase chain reaction detection of ropy Pediococcus damnosus strains in wine.

Glucan-producing strains of Pediococcus damnosus are considered as spoilage micro-organisms because synthesis of glucan leads to an unacceptable viscosity of wine. In this report, we present a polymerase chain reaction (PCR) procedure to detect the presence of such strains in wines. We developed a direct DNA isolation method from the wine microflora using polyvinylpyrrolidone in order to decrease the polyphenolic concentration. The sequence of the plasmid involved in glucan production allowed the design of a primer pair usable for a specific and sensitive PCR procedure, leading to the amplification of a 563-bp fragment. The detection limit in wine was 102 cfu ml-1. The detection sensitivity could be increased by using a second primer pair in nested PCR assays. The method proved to be efficient for the early and sensitive detection of ropy Ped. damnosus strains during wine-making. Time-consuming culture and colony isolation steps are no longer needed.

A rapid DNA isolation procedure for the identification of Campylobacter jejuni by the polymerase chain reaction.

We have developed an efficient process for rapidly isolating campylobacter DNA using mechanical disruption combined with the guanidine-based reagent DNAzol. Template DNA was isolated by this method from cultures of Campylobacter jejuni resistant to lysis by boiling or enzymes and identified following polymerase chain reaction (PCR) amplification using primers specific for the hippuricase gene. Direct detection of campylobacters in poultry-processing samples by PCR is demonstrated in chicken carcass rinses spiked with lysis-resistant C. jejuni. Our results indicate that this method of DNA isolation may be ideal for direct PCR detection of pathogenic bacteria in complex samples of widely varied origin, especially when the target organisms are difficult to lyse by other means.

Direct Polymerase Chain Reaction Detection of Campylobacter jejuni and Campylobacter coli in Raw Milk and Dairy Products

[This corrects the article on p. 3534 in vol. 59.].

Direct polymerase chain reaction detection of Campylobacter jejuni and Campylobacter coli in raw milk and dairy products.

A polymerase chain reaction (PCR) method designed to sensitively detect and identify Campylobacter jejuni and Campylobacter coli without the need for isolating and culturing strains is described. The intergenic sequence between the flagellin genes flaA and flaB was amplified and characterized with a triple primer or seminested primer approach. A total of 50 bacterial strains, 27 of C. jejuni and C. coli and 23 of other species, were tested, giving no false-positive or false-negative results. The detection limit as determined by ethidium bromide staining of amplification products on agarose gels was 10 bacteria or less in artificially contaminated water, milk, and soft cheese samples with the seminested primer PCR assay. As an application of the PCR system, a set of 93 samples of milk and other dairy products was screened for the presence of C. jejuni and C. coli. We identified six positive samples (6.5%), while none were found with a conventional culture method.

Detection of mycoplasma bovis using in vitro deoxyribonucleic acid amplification

Detection of Mycoplasma bovis by traditional culture methods is rather time-consuming and is often hampered by bacterial contamination. The development of a rapid and specific alternative is, therefore, an important prerequisite in improving the diagnosis of bovine diseases caused by this agent. The authors have successfully used nucleic acid probes containing genomic restriction fragments of M. bovis cloned into the plasmid vector pUC19 for species-specific detection by dot blot hybridisation of small quantities of M. bovis deoxyribonucleic acid (DNA). The problem of direct M. bovis detection from contaminated milk could not be solved using this procedure. Therefore, further research was conducted using in vitro DNA amplification by polymerase chain reaction (PCR). Species-specific nucleic acid probes were sequenced and suitable PCR primers selected. Using the PCR procedure, ten colony-forming units (CFU) were detected from broth cultures and, after DNA isolation, the equivalent of 1 CFU was detected. Direct detection of M. bovis from biological samples proved extremely difficult due to protein interference. It was shown, however, that direct PCR detection from milk is possible after effective protein removal by combined extraction and protease digestion.