PCR Assay For Identification Research Articles

Diagnosis of HEV infection by serological and real-time PCR assays: a study on acute non-A-C hepatitis collected from 2004 to 2010 in Italy

BackgroundThe impact of hepatitis E in developed countries, like Italy, still requires a clear definition. In the present study, we evaluated HEV infection in patients with acute non-A-C hepatitis by an approach comparing data from Real-time PCR and serological assays.MethodsIn a first analysis, sera from 52 patients hospitalized with a diagnosis of acute viral non-A-C hepatitis in Italy were tested by in-house Real-Time PCR assay for identification of Hepatitis E Virus (HEV) RNA and by anti-HEV IgM and IgG assays. In a subsequent analysis, selected samples were evaluated by additional IgM tests to confirm diagnosis.ResultsAmong the 52 samples, 21 showed positive results for all three markers (IgM, IgG and HEV RNA). One patient showed HEV RNA as single marker. Uncertain results were found in 8 samples while the remaining 22 were negative for all markers. Further analysis of the 8 undefined samples by additional IgM tests confirmed HEV infection in 1 patient. Overall, acute HEV infections were reliably identified in 23 (44.2%) out of 52 patients.ConclusionsIn the present paper, we performed a study evaluating HEV infection in 52 sporadic non-A-C acute hepatitis cases. All samples were collected from 2004 to 2010 in Italy. By a diagnostic strategy based on genomic and serological assays we identified HEV infections in 23 out of 52 patients (44.2%), a percentage higher than previous estimates. Thus, the actual impact of HEV infections in Italy needs to be further evaluated on a national scale by a diagnostic strategy based on multiple and last generation assays.

A robust PCR primer design platform applied to the detection of Acidobacteria Group 1 in soil.

Environmental biosurveillance and microbial ecology studies use PCR-based assays to detect and quantify microbial taxa and gene sequences within a complex background of microorganisms. However, the fragmentary nature and growing quantity of DNA-sequence data make group-specific assay design challenging. We solved this problem by developing a software platform that enables PCR-assay design at an unprecedented scale. As a demonstration, we developed quantitative PCR assays for a globally widespread, ecologically important bacterial group in soil, Acidobacteria Group 1. A total of 33 684 Acidobacteria 16S rRNA gene sequences were used for assay design. Following 1 week of computation on a 376-core cluster, 83 assays were obtained. We validated the specificity of the top three assays, collectively predicted to detect 42% of the Acidobacteria Group 1 sequences, by PCR amplification and sequencing of DNA from soil. Based on previous analyses of 16S rRNA gene sequencing, Acidobacteria Group 1 species were expected to decrease in response to elevated atmospheric CO2. Quantitative PCR results, using the Acidobacteria Group 1-specific PCR assays, confirmed the expected decrease and provided higher statistical confidence than the 16S rRNA gene-sequencing data. These results demonstrate a powerful capacity to address previously intractable assay design challenges.

Rapid identification of <I>Streptococus agalactiae</I> and <I>Streptococus iniae</I> with duplex PCR assay

Streptococus agalactiae and S. iniae are two common pathogens of fish Streptococcus diseases. Symptoms of disease caused by both Streptococcus pathogens were quite similar, and it was difficult to distinguish the two pathogens from each other directly from the symptoms. Therefore it is necessary to develop a rapid identification method in order to control the disease effectively and rapidly. According to the cfb gene sequence of S. agalactiae and 16S rRNA gene sequence of S. iniae, two pairs of primers were designed and synthesized, and a rapid duplex PCR assay for identification of S. agalactiae and S. iniae was established by optimization of amplification conditions. A 474 bp fragment specific for S. agalactiae and a 296 bp fragment for S. iniae were produced, but no product was amplified from the other common pathogenic bacteria of fish such as Aeromonas hydrophila and Pseudomonas aeruginosa. The method is quite sensitive, and can detect a template concentration as low as 3.2×10-3 ng/μL for S. agalactiae and 3.0×10-2 ng/μL for S. iniae, respectively. A total of eleven pathogen samples collected from pond-cultured tilapia in Guangdong and Hainan provinces were analyzed by duplex PCR. Sequencing and BLAST analysis revealed that all the sequences were cfb gene of S. agalactiae. Results of molecular identification were consistent with the previous results of biochemical assays. The double PCR method would provide a sensitive and accurate method for rapid identification of S. agalactiae and S. iniae.

PP-040 Real-time PCR assay for identification of Brucella abortus in bulk milk samples in Iran

PP-040 Real-time PCR assay for identification of Brucella abortus in bulk milk samples in Iran

An effective sequence characterized amplified region‐PCR method derived from restriction site‐amplified polymorphism for the identification of femaleSchistosoma japonicumof zoonotic significance

In the present study, restriction site-amplified polymorphism (RSAP) markers were used to examine the genetic variability of Schistosoma japonicum isolates from different endemic provinces in mainland China. Of the 45 pairs of primers screened, 10 RSAP markers showed a clear banding pattern with good resolution; however, only six exhibited a polymorphism among different isolates. Among six RSAP markers, one pair of primers (R8+R10) was able to differentiate male and female parasites, and amplified one constant specific band for female S. japonicum isolates. The specific band was recovered, re-amplified and sequenced, and a sequence of 162 bp was obtained. Based on this sequence, a pair of specific primers was designed and used to develop sequence characterized amplified region (SCAR)-PCR assay for identification and differentiation of female S. japonicum isolates. The SCAR-PCR assay allowed the specific identification of female S. japonicum, with no amplicons being amplified from male S. japonicum, Fasciola hepatica, Clonorchis sinensis, S. mansoni (male and female parasite). DNA sequencing confirmed the identity of the amplified products. The minimum amount of DNA detectable using SCAR-PCR assay was 0.3 ng for female S. japonicum. The SCAR-PCR was able to differentiate effectively the male and female S. japonicum worms collected from 12 geographical origins in eight endemic provinces, the gender of which was known based on the morphological and biological features. These results showed that SCAR-PCR provides an effective tool for the sex differentiation studies of S. japonicum, identification of female S. japonicum, diagnosis and epidemiological survey of S. japonicum infections in animals and human.

Using of PCR assay for identification of Helicobacter Species in hens\ eggs

The prevalence of Helicobacter Species in hens\\\\\\\' eggs sold in Assiut and Qena cities, Egypt was determined. A total of 300 hens\\\\\\\' eggs were collected from poultry farms and farmers, houses from both cities in which every 5 eggs were represented as one egg pooled sample. Each of egg shell and content was subjected to procedures of isolation of Helicobacter Species followed by PCR assay for the 16S rRNA gene for identification. The obtained results revealed that H. pylori recorded as the highest percentage of contamination (23.33%) followed by H. pullorum (20%). Contamination of egg shells (18.3%) and contents (11.6%) in farmer\\\\\\\'s houses was higher than poultry farm ones, concluding that egg shell was more subjected to contamination with Helicobacter Species than egg content. All H. pylori and H. pullorum isolates were confirmed by PCR by detection of one PCR product on agarose gel at 109 bp in case of H. pylori and at 477 bp in case of H. pullorum that corresponding to 16S rRNA region of the gene, confirming the specificity of PCR assay for identification of both strains. Regarding the in vitro susceptibility testing, it was found that both strains were sensitive to almost antibiotic used except for ceftriaxone and Sulphamethoxazole +Trimethoprim.

Multiplexed Reverse Transcriptase PCR Assay for Identification of Viral Respiratory Pathogens at the Point of Care

Multiplexed Reverse Transcriptase PCR Assay for Identification of Viral Respiratory Pathogens at the Point of Care

Biologic diversity of polyomavirus BK genomic sequences: Implications for molecular diagnostic laboratories

Data on polyomavirus genomic diversity has greatly expanded in the past few years. The implications of viral DNA sequence variation on the performance of molecular diagnostic assays have not been systematically examined. 716 BK, 1626 JC, and 73 SV40 virus sequences available in GenBank were aligned using Clustal-X. Five different published BKV PCR assays currently in use at major medical centers were evaluated for primer and probe mismatches with available GenBank sequences. Coverage of naturally occurring BKV strains varied amongst different assay methods. Targeted viral sequences showed major mismatch with primer or probe sequence in up to 30.7% of known BKV strains. BKV subtypes IVa, IVb, and IVc were more prone to this problem, reflecting common use of Type I Dun sequence for assay design. Despite the known polymorphism of this gene, 484 VP-1 sequences with conserved areas potentially suitable for PCR assay design are available. Assay targets in the Large T-antigen and agnogene are less subject to genetic variation, but sequence information corresponding to the latter two genes is available only for 164 and 174 published strains, respectively. Cross reactivity of appropriately selected BKV primers with JCV and SV40 sequences available in current databases was not a significant problem.

Sensitive and Broadly Reactive Reverse Transcription-PCR Assays To Detect Novel Paramyxoviruses

We have developed a set of reverse transcription-PCR assays for the detection and identification of known and novel paramyxoviruses in clinical specimens. Primers were designed from the conserved motifs of the polymerase pol gene sequences to detect members of the Paramyxovirinae or Pneumovirinae subfamily or groups of genera within the Paramyxovirinae subfamily. The consensus-degenerate hybrid oligonucleotide primer design and seminested or nested PCR assay design were used to enhance the breadth of reactivity and sensitivity of the respective assays. Using expressed RNA and 10-fold dilution series of virus-infected tissue culture isolates from different members of the family or genera, these assays were able to detect on average between 100 and 500 copies of template RNA. The assays were specific to the respective group of genera or subfamily viruses. This set of primers enhances our ability to look for novel viruses in outbreaks and diseases of unknown etiology.

Development of a multiplex quantitative fluorescent PCR assay for identification of rearrangements in the AZFb and AZFc regions

The azoospermia factor b (AZFb) and azoospermia factor c (AZFc) regions in the human Y chromosome consist of five palindromes constructed from six distinct families of amplicons and are prone to rearrangement. Partial deletion and duplication in the region can cause azoospermia or oligozoospermia and male infertility. The aim of the study was to establish a quantitative fluorescent PCR (QF-PCR) assay to classify AZFb and AZFc rearrangements. A single pair of fluorescent primers was designed to amplify simultaneously the amplicon in AZFc and the length-variant homologous sequences outside of the region as control. Since the copy number of the control sequences is fixed in the human genome, dosage of the target could be easily obtained through comparing the height of the fluorescent peaks between the target and the control after amplification with limited PCR cycles. Most types of rearrangements in AZFb and AZFc regions could be classified with QF-PCR containing four such primer pairs. Eleven types of rearrangement in AZFb and AZFc regions were well discriminated with QF-PCR. In conclusion, QF-PCR is a simple and reliable method to detect rearrangements in AZFb and AZFc.

Predicting the sensitivity and specificity of published real-time PCR assays

BackgroundIn recent years real-time PCR has become a leading technique for nucleic acid detection and quantification. These assays have the potential to greatly enhance efficiency in the clinical laboratory. Choice of primer and probe sequences is critical for accurate diagnosis in the clinic, yet current primer/probe signature design strategies are limited, and signature evaluation methods are lacking.MethodsWe assessed the quality of a signature by predicting the number of true positive, false positive and false negative hits against all available public sequence data. We found real-time PCR signatures described in recent literature and used a BLAST search based approach to collect all hits to the primer-probe combinations that should be amplified by real-time PCR chemistry. We then compared our hits with the sequences in the NCBI taxonomy tree that the signature was designed to detect.ResultsWe found that many published signatures have high specificity (almost no false positives) but low sensitivity (high false negative rate). Where high sensitivity is needed, we offer a revised methodology for signature design which may designate that multiple signatures are required to detect all sequenced strains. We use this methodology to produce new signatures that are predicted to have higher sensitivity and specificity.ConclusionWe show that current methods for real-time PCR assay design have unacceptably low sensitivities for most clinical applications. Additionally, as new sequence data becomes available, old assays must be reassessed and redesigned. A standard protocol for both generating and assessing the quality of these assays is therefore of great value. Real-time PCR has the capacity to greatly improve clinical diagnostics. The improved assay design and evaluation methods presented herein will expedite adoption of this technique in the clinical lab.

MuPlex: multi-objective multiplex PCR assay design

We have developed a web-enabled system called MuPlex that aids researchers in the design of multiplex PCR assays. Multiplex PCR is a key technology for an endless list of applications, including detecting infectious microorganisms, whole-genome sequencing and closure, forensic analysis and for enabling flexible yet low-cost genotyping. However, the design of a multiplex PCR assays is computationally challenging because it involves tradeoffs among competing objectives, and extensive computational analysis is required in order to screen out primer-pair cross interactions. With MuPlex, users specify a set of DNA sequences along with primer selection criteria, interaction parameters and the target multiplexing level. MuPlex designs a set of multiplex PCR assays designed to cover as many of the input sequences as possible. MuPlex provides multiple solution alternatives that reveal tradeoffs among competing objectives. MuPlex is uniquely designed for large-scale multiplex PCR assay design in an automated high-throughput environment, where high coverage of potentially thousands of single nucleotide polymorphisms is required. The server is available at .

T-cell therapy targeting minor histocompatibility Ags for the treatment of leukemia and renal-cell carcinoma.

healthsciences The graft-versus-host (GvH) and graft-versus-leukemia (GvL) reactions that occur after MHC-identical allogeneic hematopoietic cell transplantation (HCT) are mediated by T lymphocytes derived from the donor. We have previously shown that CD8 1 cytotoxic T lymphocytes (CTL) specific for minor histocompatibility (H) Ags expressed in recipient hematopoietic cells, including leukemic cells, but not widely expressed in non-hematopoietic tissues, can be isolated from most MHC-identical transplant recipients [1]. These CTL lyse AML and ALL blasts in vitro, and can inhibit the engraftment of human AML stem cells in nonobese diabetic (NOD)/SCID mice [2]. CD8 1 CTL clones specific for tissue-restricted minor H-Ags represent potential therapeutic reagents for adoptive T-cell therapy, designed to selectively enhance the GvL effect without inducing GvHD. A Phase I trial has been initiated at the Fred Hutchinson Cancer Research Center to evaluate the safety and antileukemic efficacy of adoptive T-cell therapy with CD8 1 CTL clones specific for tissue-restricted minor H-Ags, in patients with advanced myelodysplastic syndrome (MDS) and acute leukemia who relapse after HCT from an MHCidentical donor. The CTL clones are generated from transplant recipients prospectively at the time of transplant, and characterized for tissue specificity and Class I MHC restriction. Sequencing of each clone’s uniquely rearranged T-cell receptor b -chain V-D-J region allows the design of PCR assays that facilitate in vivo tracking of adoptively transferred CTL. Upon post-transplant relapse, patients receive cytoreductive chemotherapy, followed by three infusions of minor H-Ag-specific CTL at an escalating dose. If no toxicity is observed, additional CTL infusions are administered at weekly intervals, followed by a 14-day course of low-dose IL-2. In concurrent laboratory studies, cDNA expression cloning is being used to identify the genes encoding the Ags recognized by the CTL clones used in T-cell therapy. Graft-versus-tumor effects are also observed after nonmyeloablative MHC-identical HCT for renal-cell carcinoma (RCC), and studies to investigate the specificity of effector cells involved have recently been initiated [3]. CD8 1 minor H-Ag-specific CTL clones have been isolated from RCC patients after nonmyeloablative HCT. Several CTL clones lyse RCC cell-lines in an MHCrestricted fashion, suggesting that adoptive T-cell therapy targeting minor Ags expressed by RCC cells could be used to enhance the graft-versus-tumor effect after allogeneic HCT for metastatic RCC.