Individual PCRs Research Articles

Prevalence and Phylogenetic Analysis of Lipoprotein-Gene ragB-1 of Porphyromonas gingivalis-A Pilot Study.

Porphyromonas gingivalis (P.g.) is a key pathogen involved in periodontal diseases. The aim of this study was to investigate the prevalence and phylogenetic origin of the lipoprotein-gene ragB in its most virulent variant, ragB-1 (co-transcribed with ragA-1 as locus rag-1), in different P.g. strains collected worldwide. A total of 138 P.g. strains were analyzed for the presence of ragB-1 by pooled analysis and subsequently individual PCRs. Sequencing a core fragment of ragB-1 of the individual strains made it possible to carry out a phylogenetic classification using sequence alignment. In total, 22 of the 138 P.g. strains tested positive for ragB-1, corresponding to a prevalence of 16%. The fragment investigated was highly conserved, with variations in the base sequence detected in only three strains (OMI 1072, OMI 1081, and OMI 1074). In two strains, namely OMI 1072 (original name: I-433) and OMI 1081 (original name: I-372), which originate from monkeys, two amino-acid alterations were apparent. Since ragB-1 has also been found in animal strains, it may be concluded that rag-1 was transferred from animals to humans and that this originally virulent variant was weakened by mutations over time so that new, less virulent, adapted commensal versions of rag (rag-2, -3, and -4), with P.g. as the host, evolved.

Phylogenetic Groups/B2 Subgroup Distributions, Serogrouping and Identification of Virulence Factors in Extended-Spectrum Cephalosporin-Resistant Escherichia coli Strains Isolated from the Stool of Healthy Children Under 10 Years Old

Background: Segregation of Escherichia coli (E. coli) into the phylogenetic groups was observed in the experiments so that group B2 contained the enteropathogenic E. coli (EPEC) strains and extraintestinal pathogenic E. coli (ExPEC). Objectives: This study aimed to identify B2 phylogenetic groups in the extended-spectrum Cephalosporins resistant E. coli isolated from the stool of healthy children under 10 years old. Methods: One hundred E. coli resistant to broad-spectrum Cephalosporins were collected from the feces of healthy children under 10. Subsequently, we grouped phylogenetic via PCR based on the genes yjaA, chuA, arpA, as well, as TspE4.C2. Then, according to Clermont et al.’s study, we used two individual multiplex PCRs for identifying B2 sub-groups (I-X subgroups). Serogroup typing with the 12 O-antigen was analyzed via PCR, and finally, 10 virulence genes (cnf1, papG, ibeA, malX, usp, cdt, eae, bfp, and afa-Dr) were identified with PCR. Results: The age range of the healthy children was between 1 and 10 years. The B2 and unknown phylogroups were the most common strains in this study. The most common B2 subgroups were I (2%), IX (1%), V (8%), IV, V, VII (1%), IX, V (3%), IX, V, III, I (1%), IX, V, III, VII, I (%1), V, I (6%), V, III, I (3%), and V, III, VII (1%), with each subgroup carrying distinctive sets of ExPEC virulence markers. The results also showed that 29% of E. coli in the healthy children had malX and 23% had papGII. It was also found that 32% of the strains isolated from the healthy children had antigens O2 and 36% were unknown. Conclusions: In this study, 27% of the strains belonged to B2 phylogroup and 6% to B1 phylogroup. Moreover, serogroups O2, O16, and O25 were predominant and belonged to B2 phylogroup. Moreover, malX, papGII, usp, papGIII, aggR, and eae virulence genes also had the highest to lowest supply among the tested strains, respectively. Moreover, B2 isolates were shown to have further virulence-related genes in comparison to the non B2 isolates.

Moderate positive predictive value of a multiplex real-time PCR on whole blood for pathogen detection in critically ill patients with sepsis

A novel multiplex real-time PCR for bloodstream infections (BSI-PCR) detects pathogens directly in blood. This study aimed at determining the positive predictive value (PPV) of BSI-PCR in critically ill patients with sepsis. We included consecutive patients with presumed sepsis upon admission to the intensive care unit (ICU). The multiplexed BSI-PCR included 17 individual PCRs for a broad panel of species- and genus-specific DNA targets. BSI-PCR results were compared with a reference diagnosis for which plausibility of infection and causative pathogen(s) had been prospectively assessed by trained observers, based on available clinical and microbiological evidence. PPV and false positive proportion (FPP) were calculated. Clinical plausibility of discordant positive results was adjudicated by an expert panel. Among 325 patients, infection likelihood was categorized as confirmed, uncertain, and ruled out in 210 (65%), 88 (27%), and 27 (8%) subjects, respectively. BSI-PCR identified one or more microorganisms in 169 (52%) patients, of whom 104 (61%) had at least one detection in accordance with the reference diagnosis. Discordant positive PCR results were observed in 95 patients, including 30 subjects categorized as having an “unknown” pathogen. Based on 5525 individual PCRs yielding 295 positive results, PPV was 167/295 (57%) and FPP was 128/5525 (2%). Expert adjudication of the 128 discordant PCR findings resulted in an adjusted PPV of 68% and FPP of 2%. BSI-PCR was all-negative in 156 patients, including 79 (51%) patients in whom infection was considered ruled out. BSI-PCR may complement conventional cultures and expedite the microbiological diagnosis of sepsis in ICU patients, but improvements in positive predictive value of the test are warranted before its implementation in clinical practice can be considered.

Lack of correlation of virulence gene profiles of Staphylococcus aureus bacteremia isolates with mortality

PurposeWhole genome sequencing (WGS) analysis of Staphylococcus aureus is increasingly used in clinical practice. Although bioinformatics tools used in WGS analysis readily define the S. aureus virulome, the clinical value of this type of analysis is unclear. Here, virulence genes in S. aureus bacteremia (SAB) isolates were evaluated by WGS, with superantigens (SAgs) further evaluated by conventional PCR and functional assays, and results correlated with mortality. Methods152 SAB isolates collected throughout 2015 at a large Minnesota medical center were studied and associated clinical data analyzed. Virulence genes were identified from previously-reported WGS data (https://doi.org/10.1371/journal.pone.0179003). SAg genes sea, seb, sec, sed, see, seg, seh, sei, sej, and tst were also assessed by individual PCR assays. Mitogenicity of SAgs was assessed using an in vitro proliferation assay with splenocytes from HLA-DR3 transgenic mice. ResultsOf the 152 SAB isolates studied, 106 (69%) were methicillin–susceptible S. aureus (MSSA). The number of deaths attributed and not attributed to SAB, and 30-day survivors were 24 (16%), 2 (1%), and 128 (83%), respectively. From WGS data, both MSSA and MRSA had high proportions of adhesion (>80%) and immune-evasion (>70%) genes. There was no difference in virulomes between survivor- and non-survivor-associated isolates. Although over 60% of SAB isolates produced functional SAgs, there were no differences in the distribution or prevalence of SAg genes between survivor- and non-survivor-associated isolates. ConclusionIn this study of one year of SAB isolates from a large medical center, the S. aureus virulome, as assessed by WGS, and also for SAgs using individual PCRs and phenotypic characterization, did not impact mortality.

Site-directed mutant libraries for isolating minimal mutations yielding functional changes.

Powerful, facile new ways to create libraries of site-directed mutants are demonstrated. These include: (1) one-pot-PCR, (2) multi-pot-PCR, and (3) split-mix-PCR. One-pot-PCR uses mutant oligonucleotides to generate megaprimers in situ, and it was used to randomly incorporate 28 mutations in a gabT gene in a single reaction. In more difficult cases, multi-pot-PCR can be employed: mutant megaprimers are synthesized individually, then combined in a single mutagenesis PCR. This method was used to incorporate 14 out of 15 mutations in a pabB gene. Split-mix-PCR is a conceptually novel method for creation of site-directed mutant libraries. Separate PCRs for each mutant primer are performed, followed by pooling the products of the individual reactions. The pooled mixture is re-aliquoted into individual mutant oligonucleotide PCRs. These steps are repeated for each cycle. Split-mix-PCR results in a nearly random distribution of mutation sites, and a distribution of number-of-mutations per gene that is computable and narrow. Split-mix-PCR was applied to the directed evolution of aminodeoxychorismate synthase into anthranilate synthase, and easily allowed the determination of the fewest mutations required for introduction of novel activity.

Experimental factors affecting the robustness of DNA methylation analysis

Diverging methylation frequencies are often reported for the same locus in the same disease, underscoring the need for limiting technical variability in DNA methylation analyses. We have investigated seven likely sources of variability at different steps of bisulfite PCR-based DNA methylation analyses using a fully automated quantitative methylation-specific PCR setup of six gene promoters across 20 colon cancer cell lines. Based on >15,000 individual PCRs, all tested parameters affected the normalized percent of methylated reference (PMR) differences, with a fourfold varying magnitude. Additionally, large variations were observed across the six genes analyzed. The highest variation was seen using single-copy genes as reference for normalization, followed by different amounts of template in the PCR, different amounts of DNA in the bisulfite reaction, and storage of bisulfite converted samples. Finally, when a highly standardized pipeline was repeated, the difference in PMR value for the same assay in the same cell line was on average limited to five (on a 0–100 scale). In conclusion, a standardized pipeline is essential for consistent methylation results, where parameters are kept constant for all samples. Nevertheless, a certain level of variation in methylation values must be expected, underscoring the need for careful interpretation of data.

The restriction enzyme target approach to genotyping by sequencing (GBS).

The modified genotyping by sequencing method described here emphasizes verifying the success of each library ligation by performing individual PCRs, before preparing the pool of barcoded amplicons to be sequenced. Although this extra step might seem excessive, it will give peace of mind to the researcher knowing each individual is represented in the data set and avoid additional data imputation at the analysis stage.

Individual and Multiplex PCR Assays for the Detection of Adventitious Bovine and Porcine Viral Genome Contaminants in the Commercial Vaccines and Animal Derived Raw Materials

Animal derived raw materials such as trypsin and Fetal Bovine Serum are used in vaccine manufacturing and pose the threat of introducing animal pathogens as contaminants into the final products. Thus screening for adventitious virus/ genome is part of quality control in manufacturing of biologicals. Various in-vitro and in-vivo detection assays have been developed for the detection of potential viral contaminants in vaccines. However, these assays are expensive, time consuming, labor intensive and incomplete limiting their ability to meet the increasing demands of the biological industry. Polymerase chain reaction technology scores over the in-vitro and in-vivo assays in speed, specificity, sensitivity and robustness of detection and can replace them in regular use. In the present study, a set of multiplex and individual PCRs were developed for the detection of porcine (n=6) and bovine viral genomes (n=5). Veterinary vaccines (10), human vaccines (9), porcine typsin lots (9), and fetal bovine serum (8) were screened for adventitious viral genomes using multiplex PCRs. It was observed that 60% of veterinary vaccines, 77.7% of trypsin, and 62.5% of fetal bovine serum were contaminated with adventitious viral genomes.

An alternative method to determine the 5′ extremities of non-segmented, negative sense RNA viral genomes using positive replication intermediate 3′ tailing: Application to two members of the Paramyxoviridae family

Determining the sequence of non-segmented, negative sense RNA viral genomes is far from routine and often requires the application of several techniques. In this study, an existing method used currently just for determination of the genomic 3′ extremity was used to determine both the 3′ and 5′ sequence extremities of a Newcastle disease virus and an avian metapneumovirus. This was achieved with a single 3′ nucleotide tailing reaction of both the genomic RNA and the full length, positive sense, antigenomic RNA, followed by a single reverse transcription reaction targeted to the common polynucleotide tails, and then individual PCRs specific for each extremity using PCR primers derived from the sequence of the RT primer or from neighbouring virus sequences known previously. For each virus the method was employed separately. Sequences from both viruses were in agreement with those reported previously for other paramyxoviruses, yet one extra base at the 3′ and one extra base at the 5′ were identified for the avian metapneumovirus. In this study, importantly, the newly determined extremities maintained the complementarity known to exist between the extremities of these viruses. The method was equally successful with both viruses and can be tailored easily to function with other non-segmented, negative sense viruses through minor modification of only the primer sequences.

A molecular toolkit for population genetic investigations of the ash dieback pathogen Hymenoscyphus pseudoalbidus

SummaryThe recently described ascomycete fungus Hymenoscyphus pseudoalbidus (anamorph: Chalara fraxinea) causes the current dieback of ash (Fraxinus excelsior) in large parts of Europe. The origin of this species and its relation to the native cryptic species Hymenoscyphus albidus are still enigmatic. The spatiotemporal pattern of the epidemic is typical for an introduced invasive species. However, the presence of two cryptic species indicates that hybridization or mutation might have been involved in driving speciation in this case. In this study, we present a set of 18 polymorphic microsatellite markers to study these processes in more detail on a population genetic level. Markers were designed such that they can be amplified in three individual multiplex PCRs and analysed in two fragment analysis runs. We thoroughly tested the marker set for pairwise linkage among loci, selective neutrality and Mendelian inheritance. Additionally, the markers were applied on two large collections of isolates derived from study sites in Germany. Population genetic calculations suggested a low yet significant level of differentiation, a large genotypic diversity and a limited genetic diversity within populations. Furthermore, we present additional data concerning the phylogenetic relation between H. albidus and H. pseudoalbidus, which seems to be more distantly related to each other than expected previously.

Ten polymorphic microsatellite loci for the Atlantic Silverside, Menidia menidia

Ten polymorphic microsatellite loci were isolated from the Atlantic Silverside (Menidia menidia) in order to test hypotheses regarding the role of adaptive phenotypic variation in structuring estuarine populations along coastal North America. Loci were amplified in three multiplex panels requiring a total of four individual PCRs. All loci were highly polymorphic in individuals screened from an estuary in Nova Scotia, and none exhibited significant departures from Hardy–Weinberg equilibrium. The results suggest that these loci will be sensitive to low levels of neutral divergence among populations across M. menidia populations.

Diagnosis of viral gastroenteritis by simultaneous detection of Adenovirus group F, Astrovirus, Rotavirus group A, Norovirus genogroups I and II, and Sapovirus in two internally controlled multiplex real-time PCR assays

Background Norovirus, Rotavirus group A, Astrovirus, Sapovirus and Adenovirus serotypes 40 and 41, are common causes of gastroenteritis. Conventional diagnosis of these causative agents is based on antigen detection and electron microscopy. Objective To improve the diagnostic possibilities for viral gastroenteritis, two internally controlled multiplex real-time PCRs have been developed. Study design Individual real-time PCRs were developed and optimized for the specific detection of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, Astrovirus, Adenovirus group F and Sapovirus. Subsequently, the PCRs were combined to two multiplex PCR reactions. The multiplex assays were clinically evaluated using 239 fecal samples submitted to our laboratory over a 1-year period for the routine detection of Rotavirus and/or Adenovirus antigens using the Vikia ® Rota/Adeno test (bioMérieux, Boxtel, The Netherlands). Results In general, the multiplex real-time PCR assays showed comparable sensitivity and specificity to the individual assays. A retrospective clinical evaluation showed increased pathogen detection in samples from 14% using conventional methods to 45% using PCR. Subsequently, the assay was implemented as a routine diagnostic tool. From September 2007 up to December 2009, 486 positive results were obtained in 1570 samples (31%) analyzed. Norovirus genogroup II was found the most frequently (61.1%), followed by Adenovirus (9.9%), Rotavirus (9.3%), Astrovirus (6.0%), Norovirus genogroup I (3.3%) and Sapovirus (0.4%). Conclusions Two internally controlled multiplex real-time PCR assays for the simultaneous detection of Astrovirus, Adenovirus group F, Rotavirus, Norovirus genogroups I and II and Sapovirus have shown significant improvement in the diagnosis of viral gastroenteritis.

Multiplex PCR and quality control of Epinotia aporema granulovirus production

A specific multiplex PCR was developed for the rapid and highly sensitive quality control of the viral DNA during Epinotia aporema granulovirus (EpapGV) production. At the beginning of this work only 2.3% of the EpapGV genomic sequence was known. In order to increase the availability of specific information, the terminal sequences of the inserts of several selected clones of EpapGV genomic libraries were determined. These data comprised 8.4% of the total DNA sequence and corresponded to regions distributed throughout the genome. Based on the small fraction of known sequence available a set of 32 primers was designed, using information theory to set the basis for this study. Each pair of designed primers was initially tested in individual PCRs to assess the correct size of the expected product and the sensitivity of the amplification. The specificity was verified in multiplex PCRs, using alternatively 1-3 sets of selected 5-6 primer pairs and EpapGV DNA preparations from different sources and degrees of purity. The results indicate that the multiplex PCR could be used for quality control in the bioinsecticide production, as well as in other applications such as the detection of latent infections in E. aporema colonies, and studies related to virus distribution, vertical transmission, host range, or persistence in the field.

A statistical approach for evaluation of PCR results to improve the practical limit of quantification (LOQ) of GMO analyses (SIMQUANT)

The predominant approach for quantification of genetically modified organisms (GMO) is the application of quantitative real-time PCR. However, for a large number of processed food and feed products, this approach is unsuitable, because they contain low amounts (mass) of amplifiable DNA. Here we present a novel approach, “Single molecule quantification” (SIMQUANT) for GMO quantification of samples with extremely low amounts of DNA. The approach is based on statistics and application of multiple qualitative parallel PCRs. Here the qualitative PCRs were done using real-time PCR setup, but this is not a requirement. The difference is that the quantitative real-time PCR requires that the target copy number exceeds the absolute limit of quantification (LOQabs) and provides quantity estimates by extrapolation from a linear regressional relationship between an observed cycle threshold (Ct) value and copy numbers, while with SIMQUANT the template DNA typically contains very few, e.g., one target copy per PCR volume and the quantity is estimated on the basis of observed ratio between positive and negative individual PCRs. The components of this analysis are the numbers of test samples, the size of each sample and the outcome in number and relative ratio of positive and negative test results. The approach results in a statistical estimate of the relative GM concentration based on the probability that one or more amplifiable GM template copies are present in discrete volumes. Thus, the approach is based on the ratio of discrete volumes without or with one or more PCR-amplifiable GM target copies. The approach described here can be used reliably with more than a 100-fold improvement of the practical LOQ (LOQpract) compared to real-time quantitative PCR based on standard curves.

Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process

BackgroundSince real time PCR was first developed, several approaches to estimating the initial quantity of template in an RT-PCR reaction have been tried. While initially only the early thermal cycles corresponding to exponential duplication were used, lately there has been an effort to use all of the cycles in a PCR. The efforts have included both fitting empirical sigmoid curves and more elaborate mechanistic models that explore the chemical reactions taking place during each cycle. The more elaborate mechanistic models require many more parameters than can be fit from a single amplification, while the empirical models provide little insight and are difficult to tailor to specific reactants.ResultsWe directly estimate the initial amount of amplicon using a simplified mechanistic model based on chemical reactions in the annealing step of the PCR. The basic model includes the duplication of DNA with the digestion of Taqman probe and the re-annealing between previously synthesized DNA strands of opposite orientation. By modelling the amount of Taqman probe digested and matching that with the observed fluorescence, the conversion factor between the number of fluorescing dye molecules and observed fluorescent emission can be estimated, along with the absolute initial amount of amplicon and the rate parameter for re-annealing. The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well. An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model. The additional process is helpful in most cases where the initial primer concentration exceeds the initial probe concentration. Software for applying the algorithm to data may be downloaded at ConclusionWe present proof of the principle that a mechanistically based model can be fit to observations from a single PCR amplification. Initial amounts of amplicon are well estimated without using a standard solution. Using the ratio of the predicted initial amounts of amplicon from 2 PCRs is shown to work well even when the absolute amounts of amplicon are underestimated in the individual PCRs.

A multiplex PCR for -lactamase genes of Haemophilus influenzae and description of a new blaTEM promoter variant

To establish a multiplex PCR to detect and differentiate the beta-lactamase genes of Haemophilus influenzae. A collection of H. influenzae isolates with a range of different beta-lactamase genes were tested in parallel with individual PCRs and the multiplex assay developed in this study. The multiplex method was found to be superior to previous methods and was able to correctly detect and differentiate blaROB-1 and the variants of blaTEM present in the strains tested, including a previously unrecognized variant associated with a 54 bp insertion in the promoter region. The multiplex PCR will be a useful tool for the surveillance of beta-lactamase genes in H. influenzae.

Multiplexed tandem PCR: gene profiling from small amounts of RNA using SYBR Green detection

Multiplexed tandem PCR (MT-PCR) is a process for highly multiplexed gene expression profiling. In the first step, multiple primer pairs are added to the RNA to be analysed together with reverse transcriptase and Taq DNA polymerase. Following reverse transcription, the multiplexed amplicons are simultaneously amplified for a small number of cycles so as to avoid competition between amplicons. The reaction product is then diluted and analysed in multiple individual PCRs using primers nested inside the primers used for the multiplexed amplification. As the second PCR uses a template enriched in the amplicons of interest, the conditions can be optimized to significantly reduce ‘primer dimer’ formation allowing SYBR Green chemistry to be used for quantification. MT-PCR can be configured for as little as 10 pg RNA (equivalent to a single mammalian cell) and works well with RNA extracted from archival formalin-fixed paraffin-embedded sections. We illustrate MT-PCR with gene expression profiles of breast cancer cell lines.

Diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time RNA PCR

Background:Adequate laboratory diagnosis of human rhinoviruses (hRV) and human metapneumoviruses (hMPV) requires molecular methods as viral culture lacks sensitivity. However, setting up individual PCRs for all respiratory viruses is not practical so preferentially multiplex PCRs are used.Objectives:To develop for routine diagnosis a rapid real-time PCR assay for detection of hRV and hMPV including an internal control in a single tube multiplex reaction using probes carrying different fluorophores to discriminate targets.Study design:The multiplex real-time RNA PCR was optimized to include the internal control virus and a total of 358 respiratory samples from 239 patients taken over a one-year period were analyzed by the multiplex assay.Results:The multiplex assay with co-amplification of the internal control was as sensitive and specific as the individual assays. Application of this assay on clinical samples from 239 patients in a one-year period resulted in an incidence of hRV and hMPV of 41/239 (17.1%) and 6/239 (2.5%), respectively. Inhibition, defined as poor internal control amplification, was detected in 8 (2.2%) samples. Culture was performed on these samples and only four hRV were detected.Conclusions:This real-time PCR method enables sensitive diagnosis of these two respiratory pathogens with the potential to expand the assay as part of a full molecular respiratory viral screen.

Superantigen genes encoded by the egc cluster and SaPIbov are predominant among Staphylococcus aureus isolates from cows, goats, sheep, rabbits and poultry

In recent years several new staphylococcal enterotoxins (SEs) have been described, which currently have largely unknown frequencies of occurrence and roles in human or animal disease. One hundred and ninety-one Staphylococcus aureus isolates from cows (99), goats (39), sheep (23), rabbits (15), chickens (15) and a cat (1) were screened for SE genes sea-see, seg-seo and seq and for the tst gene encoding staphylococcal toxic shock syndrome toxin-1 using multiplex PCRs and individual PCRs for the seb and sek genes. One hundred and ten isolates tested positive for at least one of these 16 superantigen (SAg)-encoding genes. There were statistically significant differences in the frequencies of some of these SAg genes between isolates from different animals. No strain possessed either the sea or see gene. The sec gene was present in 51 isolates, the sed gene in eight and the seb gene in one. The seh gene was found in four strains and the sek and seq genes together in one isolate. The most common combinations of genes were the egc cluster, bearing the seg, sei, sem, sen and seo genes, in 47 isolates, the sec, sel and tst gene combination typical of the SaPIbov pathogenicity island in 44 isolates, the egc cluster lacking the seg gene in 11 isolates, the sed and sej genes in nine isolates, and the sec and tst genes without the sel gene in seven isolates. The higher frequencies of the sec and tst genes together and the lower frequencies of the egc gene cluster among the SAg gene-positive sheep or goat isolates compared to bovine isolates were statistically significant. Of 36 bovine isolates that were mitogenic for human T lymphocytes, four were negative for the 16 SAg genes tested for, while a further 14 gave borderline results in the mitogenicity assay, 12 of which were SAg gene-negative. Twenty-nine strains lacking all the SAg genes did not induce T-cell proliferation. This survey indicates that novel SE genes seg, sei, sel, sem, sen and seo along with the sec and tst genes predominate in S. aureus from animal hosts. The mitogenicity assays indicate that further uncharacterized SAgs may be present in bovine isolates.