Real-time Polymerase Chain Reaction Platform Research Articles

Electroactive hydrolysis probe-based portable PCR platform for sequence-specific detection of nontyphoidal Salmonella drug resistance gene

Etaq-PCR, a portable real-time polymerase chain reaction platform, has been developed based on an electroactive hydrolysis probe design and immobilization-free electrochemical readout. The Etaq-PCR platform integrates cost effective commercial instruments and thermally stable cycling into a minimal device enabling real-time monitoring of PCR amplification. No complex electrode surface treatment or micro-fabrication is involved in the construction of the Etaq-PCR platform. Thus, one can easily construct a portable PCR platform via our design. Etaq-PCR achieved sufficient sensitivity, selectivity, and rapidness to meet the WHO ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid, and robust, equipment-free, and deliverable) for point-of-care diagnosis systems. It showed good tolerance to clinical sample matrix as it displayed 100% accuracy in a validation experiment testing a drug resistance gene of nontyphoidalSalmonella in 25 raw clinical isolated samples in comparison with the gold standard qPCR. All in all, Etaq-PCR is a portable, deployable, and cost-effective PCR platform suitable for clinical applications.

Effects of SNVs in ABCA1, ABCG1, ABCG5, ABCG8, and SCARB1 Genes on Plasma Lipids, Lipoproteins, and Adiposity Markers in a Brazilian Population.

Several proteins are involved in cholesterol homeostasis, as scavenger receptor class B type I and ATP-binding cassette (ABC) transporters including ABCA1, ABCG1, ABCG5, and ABCG8. This study aimed to determine the effects of single nucleotide variants (SNVs) rs2275543 (ABCA1), rs1893590 (ABCG1), rs6720173 (ABCG5), rs6544718 (ABCG8), and rs5888 (SCARB1) on plasma lipids, lipoproteins, and adiposity markers in an asymptomatic population and its sex-specific effects. Volunteers (n = 590) were selected and plasma lipids, lipoproteins, and adiposity markers (waist-to-hip and waist-to-height ratios, lipid accumulation product and body adiposity index) were measured. Genomic DNA was isolated from peripheral blood cells according to the method adapted from Gross-Bellard. SNVs were detected in the TaqMan® OpenArray® Real-Time polymerase chain reaction platform and data analyses were performed using the TaqMan® Genotyper Software. The rs2275543*C point to an increase of high-density lipoprotein size in females while in males very-low-density lipoprotein, cholesterol, and triglycerides were statistically lower (P value < 0.05). The rs1893590*C was statistically associated with lower apolipoprotein A-I levels and higher activities of paraoxonase-1 and cholesteryl ester transfer protein (P value < 0.05). The rs6720173 was statistically associated with an increase in cholesterol and low-density lipoprotein cholesterol in males; moreover, rs6544718*T reduced adiposity markers in females (P value < 0.05). Regarding the rs5888, a decreased adiposity marker in the total population and in females occurred (P value < 0.05). Multivariate analysis of variance showed that SNVs could influence components of high-density lipoprotein metabolism, mainly through ABCG1 (P value < 0.05). The ABCA1 and ABCG5 variants showed sex-specific effects on lipids and lipoproteins, while SCARB1 and ABCG8 variants might influence adiposity markers in females. Our data indicate a possible role of ABCG1 on HDL metabolism.

Comparison of epidermal growth factor receptor mutation detection turnaround times and concordance among real-time polymerase chain reaction, high-throughput next-generation sequencing and the Biocartis Idylla™ platforms in non-small cell lung carcinomas

ObjectivesActivating mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene of non-small cell lung carcinomas (NSCLC) can be targeted by the tyrosine kinase inhibitors. A number of molecular diagnostic platforms are used to detect actionable targets in the exon(s) 18, 19, 20, and 21 of the EGFR gene. The Idylla™ system (Biocartis, Mechelen, Belgium) is a relatively novel technique and is unique in integrating both sample processing and real-time polymerase chain reaction (RT-PCR) in a single cartridge. We sought to conduct this study to compare the turnaround time (TAT) and concordance of Idylla™ system with the conventional RT-PCR and next-generation sequencing (NGS) for EGFR mutation detection. MethodsIn this retrospective analysis, 38 formalin-fixed, paraffin-embedded NSCLC tissue blocks with known NGS results by Ion Torrent™ S5 NGS platform were retested by the RT-PCR and Idylla™ platforms. ResultsA total of 15 of 38 (39.4 %) tumors that showed various EGFR mutations by NGS and conventional RT-PCR techniques were subjected to the Idylla™testing. These cases satisfied the specimen adequacy criteria of at least 10 % tumor cells for the testing. The mutations detected by the NGS were also detected by the Idylla™ testing. However, NGS identified additional 3 mutations in 3 cases, involving T709 V (exon 18, n = 1) and V774 M (exon 20, n = 2). The tumors with wild type EGFR on NGS did not have any actionable mutation detected by the Idylla™. Average EGFR testing TAT by Idylla™ was only 7.2 h (4−12 hours), as compared to conventional RT-PCR taking 54 h (31−79 hours) and NGS requiring 10.7 days (7.1−14 days). The actual procedure time by conventional RT-PCR was 24 h, NGS was 6.5 days, and Idylla™ was only 3 h. ConclusionsIn summary, the Idylla™EGFR testing is an efficient, rapid, and fairly simple tool that can be used in the routine molecular laboratory with limited expertise and infrastructure and using the lowest amount of tissue material.

Latent Class Analysis for the Diagnosis of Clostridioides difficile Infection.

Clostridioides difficile infection (CDI) is an opportunistic disease that lacks a gold-standard test. Nucleic acid amplification tests such as real-time polymerase chain reaction (PCR) demonstrate an excellent limit of detection (LOD), whereas antigenic methods are able to detect protein toxin. Latent class analysis (LCA) provides an unbiased statistical approach to resolving true disease. A cross-sectional study was conducted in patients with suspected CDI (N = 96). Four commercial real-time PCR tests, toxin antigen detection by enzyme immunoassay (EIA), toxigenic culture, and fecal calprotectin were performed. CDI clinical diagnosis was determined by consensus majority of 3 experts. LCA was performed using laboratory and clinical variables independent of any gold standard. Six LCA models were generated to determine CDI probability using 4 variables including toxin EIA, toxigenic culture, clinical diagnosis, and fecal calprotectin levels. Three defined zones as a function of real-time PCR cycle threshold (Ct) were identified using LCA: CDI likely (>90% probability), CDI equivocal (<90% and >10%), CDI unlikely (<10%). A single model comprising toxigenic culture, clinical diagnosis, and toxin EIA showed the best fitness. The following Ct cutoffs for 4 commercial test platforms were obtained using this model to delineate 3 CDI probability zones: GeneXpert®: 24.00, 33.61; Simplexa®: 28.97, 36.85; Elite MGB®: 30.18, 37.43; and BD Max™: 27.60, 34.26. The clinical implication of applying LCA to CDI is to report Ct values assigned to probability zones based on the commercial real-time PCR platform. A broad range of equivocation suggests clinical judgment is essential to the confirmation of CDI.

Fast and reliable detection of carbapenemase genes in various Gram negatives using a new commercially available fluorescence-based real-time polymerase chain reaction platform

The Revogene® Carba C assay is a real-time polymerase chain reaction–based assay that runs on the microfluidic Revogene platform. It was recently designed for the detection of genes encoding the 5 major carbapenemases (NDM, VIM, IMP, KPC, and OXA-48) from various Gram-negatives. A total of 145 clinical Gram-negative strains (96 carbapenemase producers and 49 non–carbapenemase producers) were tested. The overall sensitivity and specificity were 100%. All strains co-producing double carbapenemases have been correctly detected. All non–carbapenemase producers and nontargeted carbapenemase producers gave a negative result. The sample preparation was easy to handle, taking around 5 to 10 min per isolate, with a run time of approximately 70 min. This assay is a rapid, easy-to-perform, reliable tool to detect the most common carbapenemases, with excellent sensitivity and specificity regardless of the host bacteria. Given its user friendliness, simplicity, and short time to result, the Revogene® Carba C assay is suitable for microbiology laboratories.

Rapid identification of Babesia canis and Babesia gibsoni (Asian genotype) in canine blood samples using a customized portable real-time PCR analyzer and TaqMan-based assay

Canine babesiosis is a serious infectious disease in subtropical and tropical regions. Typically, clinical detection of canine babesiosis is performed by blood smear observation or the traditional polymerase chain reaction (PCR). Herein, we developed a new TaqMan-based real-time PCR assay combined with a customized portable real-time PCR platform for a rapid and accurate detection of canine babesiosis. Two new primer/probe pairs (B18S and BITS1) were designed based on 18S ribosomal RNA and an internal transcribed spacer 1 (ITS1) sequence to differentiate Babesia canis and B. gibsoni (Asian genotype) DNAs from canine blood samples. Additionally, a corresponding customized compact real-time PCR platform with low 6-carboxyfluorescein fluorescence detection (≤5 nM), including a fast and accurate thermal cycling ability with a user-friendly interface for thermal control and data analysis, was designed for the limited space use. Both assays (B18S and BITS1) demonstrated a sensitivity of 100 copies/reaction based on the 95 % confidence interval evaluation method. The self-developed customized portable real-time PCR analyzer presented high repeatability and reproducibility with the TaqMan-based assay. Moreover, 501 clinical specimens were collected for evaluating the performance of the proposed PCR. The positive and negative predictive values were 90 % (18 of 20) and 100 % (226 of 226), respectively, for samples suspected with B. canis infection and 98 % (55 of 56) and 100 % (199 of 199), respectively, for samples suspected with B. gibsoni infection.

Genotype and Allele Frequency of P450 Oxidoreductase *28 Gene Polymorphism in South Indian Population

Objective: To establish the normative frequency of POR *28 genetic variant in South Indian population. Methods: This study is a genetic epidemiological study conducted on healthy volunteers belonging to south India. A total of 108 healthy volunteers who satisfied the eligibility criteria were included in the study. The DNA isolation was done using the QIAGEN Blood DNA isolation kit (Qiagen). The genotyping for POR *28 polymorphism was done using TaqMan genotyping master mix kits (Applied Biosystems) in real-time polymerase chain reaction platform. The data were analyzed using Fisher's exact test. P

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia.

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America.

Association of claudin2 and PRSS1‐PRSS2 polymorphisms with idiopathic recurrent acute and chronic pancreatitis: A case–control study from India

Gene polymorphisms, including those recently described in the claudin2 gene, have been implicated in recurrent acute (RAP) and chronic pancreatitis (CP). In India, RAP and CP have been associated with SPINK1 polymorphism. In this study, we evaluated the association of claudin2 and PRSS1-PRSS2 polymorphisms with idiopathic RAP and CP. We included 101 prospectively followed patients with documented idiopathic RAP (IRAP) and 96 patients who presented with idiopathic chronic pancreatitis (ICP) without previous history of AP. Controls were 156 unrelated individuals undergoing master health check or with non-specific symptoms. All the samples were genotyped for the SNPs rs7057398 in the claudin2 (CLDN2) gene and rs10273639 in the PRSS1 gene on Realtime polymerase chain reaction platform. Clinical data pertaining to patient and disease characteristics were recorded. Claudin2 and PRSS1 polymorphisms were seen in a significantly higher proportion of female patients (P = 0.01 and 0.039, respectively). Thirty-three (32.7%) patients with IRAP developed features of early CP during follow-up (mean [95% confidence interval, CI] duration of 11.3 [8.9-13.7] months). Female patients with claudin2 (rs7057398) CC genotype were at significantly higher risk for IRAP (odds ratio [OR] [95% CI] 6.75 [1.82-23.67]; P = 0.004) and progression from IRAP to CP (OR [95% CI] 7.05 [1.51-33.01]; P = 0.007). CT genotype of PRSS1 (rs10273639) was associated IRAP (OR [95% CI] 2.59 [1.1-6.13]; P = 0.030), and both CT and CC genotypes with ICP in women (OR [95% CI] 2.86 [1.12-7.31]; P = 0.033 and 3.73 [1.03-13.59]; P = 0.048, respectively). In this study, we have demonstrated the association of claudin2 (rs7057398) polymorphism with IRAP and progression of IRAP to CP, and PRSS1 (rs10273639) polymorphism with IRAP and ICP.

Dataset for genotyping validation of cytochrome P450 2A6 whole-gene deletion (CYP2A6*4) by real-time polymerase chain reaction platforms.

This data article contains a supplementary figure and validation data relating to the research article entitled “Genotyping of wild-type cytochrome P450 2A6 and whole-gene deletion using human blood samples and a multiplex real-time polymerase chain reaction method with dual-labeled probes” (Shimizu et al., Clinica Chimica Acta 441, 71–74, 2015), which presents a multiplex real-time polymerase chain reaction method with dual-labeled probes for human P450 2A6 wild-type and whole-gene deletion. Real-time methods have dramatically improved the speed of complex genetic diagnostics compared to conventional assays based on restriction enzyme digestion. Here, we show the basic assay validation data by single and multiplex determinations in comparison with commercial TaqMan copy number assays for P450 2A6.

Development and Validation of LNA-Based Quantitative Real-Time PCR Assays for Detection and Identification of the Root-Knot Nematode Meloidogyne enterolobii in Complex DNA Backgrounds.

Meloidogyne enterolobii is a quarantine root-knot nematode posing a major threat to agricultural production systems worldwide. It attacks many host plants, including important agricultural crops, ornamentals, and trees. M. enterolobii is a highly virulent and pathogenic root-knot nematode species, able to reproduce on plants resistant to other Meloidogyne spp. Significant crop damage has been reported in Asia, South America, Africa, the United States, France, and greenhouses in Switzerland. To identify potential introduction pathways and ensure appropriate phytosanitary measures and management strategies, accurate detection and identification tools are needed. Therefore, two real-time quantitative polymerase chain reaction (PCR) assays based on the second intergenic spacer region of the ribosomal DNA cistron and the cytochrome oxidase c subunit I (COI) gene using locked nucleic acid probes were developed and validated for fast and reliable detection and identification of M. enterolobii. Analytical specificity was confirmed with 16 M. enterolobii populations, 16 populations of eight closely related Meloidogyne spp., and four species from other nematode genera. Optimizing and testing the assays on two real-time PCR platforms revealed an analytical sensitivity of one juvenile in a background of 1,000 nematodes and the intended limit of detection of one juvenile per 100 ml of soil. Both assays performed equally well, with the COI-based assay showing a slightly better performance concerning detection of M. enterolobii target DNA in complex DNA backgrounds.

Comparative study of epidermal growth factor receptor mutation analysis on cytology smears and surgical pathology specimens from primary and metastatic lung carcinomas

The detection of epidermal growth factor receptor (EGFR) mutations on small biopsy or fine-needle aspiration samples is required to guide therapy in nonsmall cell lung cancer (NSCLC). In this study, the authors compared results from EGFR mutation testing on both cytologic smears and surgical specimens and also compared the performance of platforms using 2 different technologies (pyrosequencing and real-time polymerase chain reaction) for both specimen types. Specimens from 114 patients were divided into 2 subsets. The first subset had 60 paired cytology smears and surgical specimens, including 37 paired specimens from the same site and 23 paired specimens from different sites. The second subset consisted of nonpaired cytology smears and formalin-fixed, paraffin-embedded (FFPE) tissues (including 8 cell blocks), which were compared on the pyrosequencing and real-time polymerase chain reaction platforms. Laser-capture microscopy was used to enrich tumor in the FFPE specimens before DNA extraction. All cytology smears that were used in the study were adequate for analysis on both platforms. Comparison between smears and concurrent FFPE tissues from the same anatomic site had a concordance rate of 97%. The concordance rate between the pyrosequencing platform and the real-time polymerase chain reaction platform was 84% and 85% for FFPE tissues and cytology smears, respectively. The current results indicated that direct extraction and analysis of EGFR mutations from cytology smears can be performed successfully on both a pyrosequencing platform and a real-time polymerase chain reaction platform with results comparable to those achieved in matched surgical specimens. In fine-needle aspiration/endobronchial ultrasound samples with limited tissue, cytology smears can be important for molecular analysis. Cancer (Cancer Cytopathol) 2013;121:361-369. © 2012 American Cancer Society.

Combination of fluorescence color and melting temperature as a two-dimensional label for homogeneous multiplex PCR detection

Multiplex analytical systems that allow detection of multiple nucleic acid targets in one assay can provide rapid characterization of a sample while still saving cost and resources. However, few systems have proven to offer a solution for mid-plex (e.g. 10- to 50-plex) analysis that is high throughput and cost effective. Here we describe the combined use of fluorescence color and melting temperature (Tm) as a virtual 2D label that enables homogenous detection of one order of magnitude more targets than current strategies on real-time polymerase chain reaction platform. The target was first hybridized with a pair of ligation oligonucleotides, one of which harbored an artificial sequence that had a unique Tm when hybridized with a reporter fluorogenic probe. The ligated products were then amplified by a universal primer pair and denatured by a melting curve analysis procedure. The targets were identified by their respective Tm values in the corresponding fluorescence detection channels. The proof-of-principle of this approach was validated by genotyping 15 high-risk human papillomaviruses and 48 human single-nucleotide polymorphisms. The robustness of this method was demonstrated by analyzing a large number of clinical samples in both cases. The combined merits of multiplexity, flexibility and simplicity should make this approach suitable for a variety of applications.

A Second-Generation MicroRNA-Based Assay for Diagnosing Tumor Tissue Origin

Cancers of unknown primary origin (CUP) constitute 3%-5% (50,000 to 70,000 cases) of all newly diagnosed cancers per year in the United States. Including cancers of uncertain primary origin, the total number increases to 12%-15% (180,000 to 220,000 cases) of all newly diagnosed cancers per year in the United States. Cancers of unknown/uncertain primary origins present major diagnostic and clinical challenges because the tumor tissue of origin is crucial for selecting optimal treatment. MicroRNAs are a family of noncoding, regulatory RNA genes involved in carcinogenesis. MicroRNAs that are highly stable in clinical samples and tissue specific serve as ideal biomarkers for cancer diagnosis. Our first-generation assay identified the tumor of origin based on 48 microRNAs measured on a quantitative real-time polymerase chain reaction platform and differentiated 25 tumor types. We present here the development and validation of a second-generation assay that identifies 42 tumor types using a custom microarray. A combination of a binary decision-tree and a k-nearest-neighbor classifier was developed to identify the tumor of origin based on the expression of 64 microRNAs. Overall assay sensitivity (positive agreement), measured blindly on a validation set of 509 independent samples, was 85%. The sensitivity reached 90% for cases in which the assay reported a single answer (>80% of cases). A clinical validation study on 52 true CUP patients showed 88% concordance with the clinicopathological evaluation of the patients. The abilities of the assay to identify 42 tumor types with high accuracy and to maintain the same performance in samples from patients clinically diagnosed with CUP promise improved utility in the diagnosis of cancers of unknown/uncertain primary origins.

Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood

Species of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5-10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.

Determining resistance to Pseudomonas syringae in tomato, a comparison of different molecular markers

Pseudomonas syringae pv. tomato (Pst) is the causal agent of bacterial speck disease in tomato. Resistance to Pst is determined by Pto, a single resistance gene that belongs to a multi-gene family clustered on chromosome 5. Pst-resistant phenotypes in cultivated tomato are determined by a semi-dominant allele of Solanum pimpinellifolium, which was introgressed into Solanum lycopersicum in the past century. Seed companies, which are continuously interested in producing resistant varieties, can benefit from genetic markers closely linked to the Pto locus in breeding programs based on marker-assisted selection. In this research, three sequence characterised amplified region markers have been developed for identification of resistant and susceptible genotypes of Solanum lycopersicum. A cleaved amplified polymorphic sequence marker has been adapted to a real-time polymerase chain reaction platform using high-resolution melting curve analysis. Application to a tomato population for breeding programs is described. Advantages and disadvantages of the different markers are discussed.

Development and Validation of a Quantitative Real-Time Polymerase Chain Reaction Classifier for Lung Cancer Prognosis

This prospective study aimed to develop a robust and clinically applicable method to identify patients with high-risk early-stage lung cancer and then to validate this method for use in future translational studies.Three published Affymetrix microarray data sets representing 680 primary tumors were used in the survival-related gene selection procedure using clustering, Cox model, and random survival forest analysis. A final set of 91 genes was selected and tested as a predictor of survival using a quantitative real-time polymerase chain reaction-based assay using an independent cohort of 101 lung adenocarcinomas.The random survival forest model built from 91 genes in the training set predicted patient survival in an independent cohort of 101 lung adenocarcinomas, with a prediction error rate of 26.6%. The mortality risk index was significantly related to survival (Cox model p < 0.00001) and separated all patients into low-, medium-, and high-risk groups (hazard ratio = 1.00, 2.82, 4.42). The mortality risk index was also related to survival in stage 1 patients (Cox model p = 0.001), separating patients into low-, medium-, and high-risk groups (hazard ratio = 1.00, 3.29, 3.77).The development and validation of this robust quantitative real-time polymerase chain reaction platform allows prediction of patient survival with early-stage lung cancer. Utilization will now allow investigators to evaluate it prospectively by incorporation into new clinical trials with the goal of personalized treatment of patients with lung cancer and improving patient survival.

Procalcitonin as a Biomarker in Respiratory Tract Infection

Serum procalcitonin (PCT) levels rapidly increase in patients with invasive bacterial disease. PCT levels increase faster than do C-reactive protein levels. Furthermore, a rapid decrease in the PCT level is supporting evidence that the source of the bacterial infection is responding to clinical management. In patients with community-acquired bacterial pneumonia, sequential PCT levels are useful as a guide to shorter courses of antimicrobial therapy. With use of emerging multiplex real-time polymerase chain reaction platforms for the detection of viral and bacterial respiratory pathogens, it should be possible to critically assess whether an elevated serum PCT level is a valid biomarker of invasive bacterial infection.

Tag–single nucleotide polymorphism–based human leukocyte antigen genotyping in celiac disease patients from northeastern Italy

We genotyped celiac disease (CD)–associated haplotypes DQ2.5, DQ8, DQ2.2, and DQ7 in 1005 CD patients from North Eastern Italy using a Tag–single nucleotide polymorphism (SNPs) approach and real time PCR platform, checking the accuracy and reliability of the method and comparing it to traditional PCR-SSP. Only 14 of 2010 chromosomes analyzed (0.7%) showed discrepancies between the Tag-SNPs real-time polymerase chain reaction (PCR) method and the PCR–single-strand polymorphism (SSP) technique, indicating a high sensitivity and specificity (ranging from 0.987 to 1 and from 0.998 to 0.999, respectively) for tagging with respect to corresponding human leukocyte antigen (HLA) alleles identified by PCR-SSP. Moreover, the overall cost of the Tag-SNPs HLA typing method was low (3 to 4 €/sample instead of 35 to 70 €/sample with commercial kits), making it suitable for mass screenings. Hence, we believe that the Tag-SNPs HLA typing could be used to complement or replace classic HLA typing in at high-risk groups, for research purposes and eventually in population screening programs.

Microfluidic platform versus conventional real-time polymerase chain reaction for the detection of Mycoplasma pneumoniae in respiratory specimens

Rapid, accurate diagnosis of community-acquired pneumonia (CAP) due to Mycoplasma pneumoniae is compromised by low sensitivity of culture and serology. Polymerase chain reaction (PCR) has emerged as a sensitive method to detect M. pneumoniae DNA in clinical specimens. However, conventional real-time PCR is not cost-effective for routine or outpatient implementation. Here, we evaluate a novel microfluidic real-time PCR platform (Advanced Liquid Logic, Research Triangle Park, NC) that is rapid, portable, and fully automated. We enrolled patients with CAP and extracted DNA from nasopharyngeal wash (NPW) specimens using a biotinylated capture probe and streptavidin-coupled magnetic beads. Each extract was tested for M. pneumoniae-specific DNA by real-time PCR on both conventional and microfluidic platforms using Taqman probe and primers. Three of 59 (5.0%) NPWs were positive, and agreement between the methods was 98%. The microfluidic platform was equally sensitive but 3 times faster and offers an inexpensive and convenient diagnostic test for microbial DNA.