Rapid Multiplex Polymerase Chain Reaction Research Articles

Rapid multiplex polymerase chain reaction for simultaneous detection of Vibrio harveyi, V. parahaemolyticus, and V. vulnificus in pacific white shrimp (Litopenaeus vannamei)

Context: A comparatively small number of species, e.g., Vibrio parahaemolyticus and V. vulnificus , cause disease in both aquatic animals and humans. V. harveyi is marine animal pathogen and rarely causes infections in humans; however, it might become a reservoir of antibiotic-resistant bacteria forms and virulence genes. Aims: 1) to develop rapid multiplex polymerase chain reaction (PCR) assay for the simultaneous detection of V. harveyi, V. parahaemolyticus , and V. vulnificus by using vhhP2, tl , and rpoS genes as the respective target genes and 2) to evaluate specificity and determined detection of multiplex PCR technique. Materials and Methods: The multiplex PCR assay was developed and evaluated for specificity on 36 isolates of V. harveyi , 30 isolates of V. parahaemolyticus , and 14 isolates of V. vulnificus , along with other species of Vibrio and non- Vibrio bacterial isolates. Sensitivity of test was described as detection limit of pathogens in lowest amount of sample (CFU/mL or CFU/g) was determined by diluted DNA extracts of the pure cultures and spiked pacific white shrimp (Litopenaeus vannamei) samples Results: This developed multiplex PCR was proved as an accurate method, which was specific for three Vibrio species. The detection limits of V. harveyi , V. parahaemolyticus , and V. vulnificus in pure cultures and spiked shrimp samples ranged 1.05-4.8 × 10 3 CFU/mL and 1.9-7 × 10 4 CFU/g, respectively. Conclusions: This rapid multiplex PCR assay can decrease amount and process of sample preparation, which was time-consuming, and had preferable accuracy. This developed technique will be suitable and useful for food-borne pathogen detection in shrimp and horizontal gene transfer study among different Vibrio species in aquatic animals.

Randomized Trial of Rapid Multiplex Polymerase Chain Reaction-Based Blood Culture Identification and Susceptibility Testing.

The value of rapid, panel-based molecular diagnostics for positive blood culture bottles (BCBs) has not been rigorously assessed. We performed a prospective randomized controlled trial evaluating outcomes associated with rapid multiplex PCR (rmPCR) detection of bacteria, fungi, and resistance genes directly from positive BCBs. A total of 617 patients with positive BCBs underwent stratified randomization into 3 arms: standard BCB processing (control, n = 207), rmPCR reported with templated comments (rmPCR, n = 198), or rmPCR reported with templated comments and real-time audit and feedback of antimicrobial orders by an antimicrobial stewardship team (rmPCR/AS, n = 212). The primary outcome was antimicrobial therapy duration. Secondary outcomes were time to antimicrobial de-escalation or escalation, length of stay (LOS), mortality, and cost. Time from BCB Gram stain to microorganism identification was shorter in the intervention group (1.3 hours) vs control (22.3 hours) (P < .001). Compared to the control group, both intervention groups had decreased broad-spectrum piperacillin-tazobactam (control 56 hours, rmPCR 44 hours, rmPCR/AS 45 hours; P = .01) and increased narrow-spectrum β-lactam (control 42 hours, rmPCR 71 hours, rmPCR/AS 85 hours; P = .04) use, and less treatment of contaminants (control 25%, rmPCR 11%, rmPCR/AS 8%; P = .015). Time from Gram stain to appropriate antimicrobial de-escalation or escalation was shortest in the rmPCR/AS group (de-escalation: rmPCR/AS 21 hours, control 34 hours, rmPCR 38 hours, P < .001; escalation: rmPCR/AS 5 hours, control 24 hours, rmPCR 6 hours, P = .04). Groups did not differ in mortality, LOS, or cost. rmPCR reported with templated comments reduced treatment of contaminants and use of broad-spectrum antimicrobials. Addition of antimicrobial stewardship enhanced antimicrobial de-escalation. NCT01898208.

Screening test for detection of newcastle disease, avian influenza and infectious bronchitis viruses using multiplex reverse transcriptase polymerase chain reaction approach

Numerous viral pathogens are circulated in the environment of commercial chicken farms that causing the difficulty in the confirmation of diagnosis. A breakthrough on the diagnosis technique is required in order to deal with multiple viral infections. Ideally, the approach should have not only high accuracy but also economical and straightforward. The objective of this research is to develop a rapid multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) diagnostic method for three common infectious viral diseases in poultry, Newcastle Disease (ND), Avian Influenza (AI) subtype H5N1 and Infectious Bronchitis (IB). This study was successful in developing and optimizing the mRT-PCR for these three pathogens in a single reaction test. Testing 67 field samples from Sukabumi district revealed the presence of several targeted viruses. Key Words : Screening Test, Multiplex RT-PCR, Newcastle Disease, Avian Influenza, Infectious Bronchitis

Multiplex TaqMan® detection of pathogenic and multi-drug resistant Salmonella

Overuse of antibiotics in the medical and animal industries is one of the major causes for the development of multi-drug-resistant (MDR) food pathogens that are often difficult to treat. In the past few years, higher incidences of outbreaks caused by MDR Salmonella have been increasingly documented. The objective of this study was to develop a rapid multiplex real-time polymerase chain reaction (PCR) assay for simultaneous detection of pathogenic and MDR Salmonella spp. A multiplex TaqMan®real-time PCR was designed by targeting the invasin virulence gene (invA), and four commonly found antibiotic resistance genes, viz. ampicillin, chloramphenicol, streptomycin and tetracycline. To avoid false negative results and to increase the reliability of the assay, an internal amplification control (IAC) was added which was detected using a locked nucleic acid (LNA) probe. In serially diluted (5ng–50fg) DNA samples, the assay was able to detect 100 genomic equivalents of Salmonella, while in a multiplex format, the sensitivity was 1000 genomic equivalents. The assay performed equally well on artificially contaminated samples of beef trim, ground beef of different fat contents (73:27, 80:20, 85:15 and 93:7), chicken rinse, ground chicken, ground turkey, egg, spinach and tomato. While the detection limit for un-enriched inoculated food samples was 104CFU/g, this was improved to 10CFU/g after a 12-h enrichment in buffered peptone water, with 100% reproducibility. The multiplex real-time assay developed in this study can be used as a valuable tool to detect MDR virulent Salmonella, thus enhancing the safety of food.

Evaluation of a Pilot Respiratory Virus Surveillance System Linking Electronic Health Record and Diagnostic Data

During the onset of 2009 pandemic influenza A (H1N1) (pH1N1), the New York City Department of Health and Mental Hygiene implemented a pilot respiratory virus surveillance system. We evaluated the performance of this pilot system, which linked electronic health record (EHR) clinical, epidemiologic, and diagnostic data to monitor influenza-like illness (ILI) in the community. Surveillance was conducted at 9 community health centers with EHRs. Clinical decision support system alerts encouraged diagnostic testing of patients. Rapid influenza diagnostic testing (RIDT) and multiplex polymerase chain reaction assay (MassTag PCR) were performed sequentially. Nine Institute for Family Health (IFH) clinics in Manhattan and the Bronx during May 26 to June 30, 2009, the pH1N1 outbreak peak. Adult and pediatric patients presenting to IFH clinics during May 26 to June 30, 2009. By using Centers for Disease Control and Prevention guidelines, we evaluated the system's completeness, sensitivity, timeliness, and epidemiologic usefulness. Of 537 ILI visits (5.7% of all visits), 17% underwent diagnostic testing. Of the 132 specimens with both a RIDT and MassTag PCR result, 90 (68%) had a MassTag PCR-identified respiratory virus, most commonly pH1N1 (n = 69; 77%). Of the 81 specimens that met the ILI case definition, 58 (72%) were positive for a respiratory virus tested for by MassTag PCR; 48 (59%) were positive for pH1N1. Ninety-four percent of ILI patients positive for pH1N1 were 45 years or younger. Sensitivity and specificity of RIDT (29% and 94%) and ILI case definition (70% and 48%) for pH1N1 were calculated using MassTag PCR as the standard. Results of RIDT took a median of 6 days. Despite low RIDT sensitivity for pH1N1 and limited timeliness, integration of EHR and diagnostic data has potential to provide valuable epidemiologic information, guide public health response, and represents a new model for community surveillance for influenza and respiratory viruses.

UGT1A1 promoter polymorphism associated with serum bilirubin level in Saudi patients with sickle cell disease

BACKGROUND AND OBJECTIVESPolymorphism in (TA)n of the UGT1A1 promoter influences bilirubin level and risk of gallstones in patients with sickle cell disease (SCD) of African descent. Modifiers of bilirubin level and gallstones in Saudi patients with SCD are not known.DESIGN AND SETTINGSPatients with SCD presenting to participating institutions between July 2009 and July 2012 were enrolled in our study.METHODSA total of 223 SCD patients were enrolled. Laboratory workup at steady state included complete blood count, reticulocytes, serum bilirubin, lactate dehydrogenase (LDH), G6PD level, and hemoglobin (Hb) electrophoresis. The (TA)n UGT1A1 promoter polymorphism and presence of α-thalassemia were also determined.RESULTSTA6/6 in the UGT1A1 promoter was identified in 189 patients (84.7%), TA7/7 in 26 (11.7%), TA5/5 in 6 (2.7%), and TA5/6 in 2 (0.9%). Increased (TA)n of the UGT1A1 promoter (P<.0001), male gender (P=.02), higher LDH (P=.001), and lower Hb level (P=.009) were associated with higher bilirubin level, while the co-inheritance of α-thalassemia (P=.003) was linked with lower bilirubin level. UGT1A1 (TA)n (P<.0001) and Hb level (P=.005) remained significant on multivariate analysis. Gallstones were more frequent in patients with TA7/7 (72%) compared to patients with TA6/6 (57%) and TA5/5 or 5/6 (37%); however, this difference was not statistically significance (P=.18). Older age (P=.0001) and absence of α-thalassemia (P=.03) were associated with higher risk of gallstones.CONCLUSION(TA)n in the UGT1A1 promoter and intensity of hemolysis modify steady-state serum bilirubin level in SCD. Co-inheritance of α-thalassemia reduces the risk of gallstones in Saudi patients with SCD.

Comparison of Clinical Manifestation and Laboratory Findings between H1N1 and Influenza B Infection

Purpose: Influenza virus is one of the most important viruses that cause the respiratory infection seasonally. In April 2009, H1N1 was detected in America and Mexico and then there was pandemic in Korea. We investigated the difference of clinical and laboratory findings between the infections of H1N1 and Influenza B. Methods: We have retrospectively studied the patients under age of 15 years who visited Inje University Sanggye Paik Hospital from August 2009 to April 2010. Evaluation for influenza infection was performed by rapid antigen test or multiplex reverse transcriptase polymerase chain reaction. Complete blood count with differential counts, C-reactive protein and chest X-ray were checked. Results: Enrolled patients were 2,226 in H1N1-infected group and 288 in influenza B-infected group. Seasonal variation was that H1N1 in autumn and winter but influenza B in spring. The male-to- female sex ratio was same as 1.23 in each group. The mean age of H1N1-infected group was higher than influenza B-infected group (P<0.001). Fever was developed similarly in both groups (P=0.114). However, cough, sputum, rhinorrhea, vomiting, diarrhea, and headache were more prevalent in influenza B infection compared to H1N1 infection (P<0.001). Pneumonia development and admission rate were higher in influenza B infection compared to H1N1 infection (P<0.001, respectively). Conclusion: Although H1N1 infection spread rapidly, H1N1 caused not so severe symptoms than influenza B. Because of the possibility that influenza epidemic will develop repeatedly in the future, we need to evaluate more about different characteristics depending on the virus subtype and prepare for them.

Updated multiplex polymerase chain reaction for detection of 16S rRNA methylases: high prevalence among NDM-1 producers

We develop a rapid (<4 h) and reliable multiplex polymerase chain reaction for screening of 16S rRNA methylase genes conferring resistance to aminoglycosides. This study particularly underlined that 16S rRNA methylases are frequently (75%) identified among Enterobacteriaceae isolates producing the carbapenemase NDM-1.

A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples

Forensic DNA typing involves a multi-step workflow. Steps include DNA isolation, quantification, amplification of a set of short tandem repeat (STR) markers, separation of polymerase chain reaction (PCR) products by capillary electrophoresis (CE) and DNA profile analysis and interpretation. With that, the process takes around 10–12h. For several scenarios it may be very valuable to speed up this process and obtain an interpretable DNA profile, suited to search a DNA database, within a few hours. For instance in cases of national security, abduction with danger of life, risk of repetition by a serial perpetrator or when custody time of suspects is limited. By a direct and rapid PCR approach we reduced the total DNA profiling time to 2–3h after which genotyping information for the 10 STR markers plus the amelogenin (AMEL) marker present in the commercially available AmpFℓSTR® SGM Plus™ (SGM+) profiling kit is obtained. This reduction in time is achieved by using the following elements: (1) the inhibitor tolerant, highly processive Phusion® Flash DNA polymerase; (2) a modified, non-adenylated allelic ladder; (3) the quick PIKO® thermal cycler system with ultra-thin walled reaction tubes; (4) profile interpretation guidelines with an increased allele calling threshold, modified stutter ratios and marked low-level artefact peaks and (5) regulation of sample input by the use of mini-tapes that lift a limited amount of cell material from swabs or fabrics. The procedure is specifically effective for high level DNA, single source samples such as samples containing saliva, blood, semen and hair roots. Success rates, defined as a complete DNA profile, depend on stain type and surface. Due to the use of tape lifting as the sampling technique, the swab or fabric remains dry and intact and can be analyzed at a later stage using regular procedures. Validation experiments were performed which showed that the protocol effectively instructs researchers unfamiliar with the procedure. We have incorporated direct and rapid PCR in a “DNA-6h” service that can assist police investigations by rapidly deriving DNA information from trace evidence left by a perpetrator, searching the STR profile against a DNA database and reporting the outcomes to police or prosecution.

SEROTYPE DISTRIBUTION OF LISTERIA MONOCYTOGENES ISOLATED FROM TURKEY MEAT BY MULTIPLEX PCR IN TURKEY

ABSTRACTThe objectives of this study were to identify the serotypes of Listeria monocytogenes isolated from packaged fresh turkey meat samples marketed in Ankara, Turkey, and to determine the seasonal distribution of the serotypes. Out of 37 L. monocytogenes isolates obtained from 180 turkey meat samples, 19 (51.4%), 10 (27.0%) and 8 (21.6%) were serotyped by multiplex polymerase chain reaction as 4b (or 4d, 4e), 1/2a (or 3a) and 1/2b (or 3b), respectively. Serotypes 1/2a and 4b were dominant in turkey meat samples collected in the summer, and 4b was the most prevalent L. monocytogenes serotype during the winter. Serotype 4b was the most prevalent serotype in turkey meat cuts and legs, whereas 1/2b was the dominant serotype in turkey breast samples. Turkey meat can be a potential risk for public health; therefore, it should be produced under appropriate hygienic and technological conditions and cooked adequately before consumption.PRACTICAL APPLICATIONSSerotyping has been widely used to characterize L. monocytogenes isolates and is important from the epidemiological point of view. Serotypes 1/2a, 1/2b, 1/2c and 4b are the predominant (&gt;95%) serotypes isolated from food samples and human listeriosis cases among 13 L. monocytogenes serotypes. Conventional agglutination and pulsed‐field gel electrophoresis methods have been routinely used for serotyping and characterizing L. monocytogenes but these techniques are today expensive and time consuming. In the present study, rapid and practical multiplex polymerase chain reaction assay was used to differentiate the major L. monocytogenes serotypes (1/2a, 1/2b, 1/2c and 4b) in turkey meat.

A simple multiplex polymerase chain reaction to determine ABO blood types of rhesus macaques (Macaca mulatta)

Rhesus macaques are the most common nonhuman primate model organism used in biomedical research. Their increasingly frequent use as subjects in studies involving transplantation requires that blood and other tissue antigens of donors and recipients be compatible. We report here an easy and rapid multiplex polymerase chain reaction (PCR) to determine the ABO blood group phenotypes of rhesus macaques that can be performed with only small amounts of DNA. We phenotyped 78 individuals and found this species to exhibit the A, B and AB phenotypes in frequencies that vary by geographic region. The probability of randomly pairing rhesus macaque donors and recipients that exhibit major ABO phenotype incompatibility is approximately 0.35 and 0.45 for Indian and Chinese rhesus macaques, respectively.

Sexing of newly-hatched chicks using DNA isolated from chorio-allantoic membrane samples by polymerase chain reaction in Denizli chicken

1. The aim of the present study was to determine the sex of newly-hatched chicks of Denizli chicken, a local Turkish breed, by polymerase chain reaction (PCR) using DNA extracted from the chorioallantoic membrane (CAM). 2. Fertilised eggs were incubated individually and a total of 20 CAM samples were collected following the hatching process. DNA was isolated from the CAM samples and PCR was performed using W-repeat (W) and 18 S ribosomal gene (R) primers. 3. Screening of the PCR products by agarose gel electrophoresis revealed that males have a single band (256 bp) and females have an extra second band (415 bp) as expected. 4. The present study describes a reliable, rapid, and simple multiplex PCR protocol that can be put into use to sex local breeds of chicken in which phenotypic sexing is impossible, using DNA isolated from the CAM that is discarded and remains attached to the egg shell following the hatching process.

New real-time PCR-based method for the joint genotyping of JAK2 (V617F) with inherited thrombophilic F5 and F2 mutations

Background During the last years the appearance of the acquired V617F mutation of the Janus Kinase 2 gene (JAK2) in patients suffering different thrombotic events has been described. We decided to develop a new and rapid multiplex real-time Polymerase Chain Reaction (PCR) in order to detect the V617F mutation together with the inherited prothrombotic mutations of factors F5 and F2. Design and methods The method was carried out on the LightCycler 2.0 (Roche Diagnostics, Mannheim, Germany) and consisted in a first step of simultaneous amplification by real-time PCR of the three genes to be genotyped, in a 20 μl closed tube, using a primer pair together with the correspondent FRET-hybridization probes for each gene. Results We assayed 41 samples in the multiplex PCR reaction, 19 were positive (46.34%) for V617F mutation. From the V617F positive samples we found 1 sample heterozygous for F2 (5.26%) and 1 sample heterozygous for F5 (5.26%), so a 10.52% of the samples tested combine V617F mutation with inherited thrombophilic mutations. Results were clear, rapid and reliable allowing a significant time saving. Conclusions The technique presented in this manuscript is a new achievement in the field of the molecular diagnosis that combines the genotyping of F5 and F2 with the assessment of the JAK2 (V617F) mutation load.

Multiplex PCR assay for dissemination and diversity of extended-spectrum beta-lactamase genes in Shigella isolates

To develop a rapid and simple multiplex polymerase chain reaction (PCR) method which discriminates extended-spectrum beta-lactamases (ESBLs) genes in sporadic Shigella isolates from 1998 to 2007 in Hangzhou city, China. After ESBLs screening according to the Clinical and Laboratory Standards Institute (CLSI) method, CTX-M, TEM, SHV and OXA-1 encoding genes were detected by using a multiplex PCR method, and the results were verified by 8 single gene PCR amplification. Seventeen isolates harbored ESBLs genes among 195 Shigella isolates (8.72%). Genes encoding CTX-M (17 strains), TEM (2 strains), OXA-1 (10 strains) and SHV (0 strains) were discriminated with multiplex PCR analysis, which coincided with eight single gene PCR analysis at 94.12%. Multiplex PCR should be a suitable tool for initial rapid screening and discriminating ESBLs genes in Shigella isolates. With similar trend of national surveillance data, the proportion of sporadic Shigella isolates harbouring ESBLs genes might probably be on increase.

RETRACTED: Allelotyping PCR for Detection and Screening of Salmonella enterica Serovar Enteritidis and Typhimurium

3 Abstract: Classical Salmonella sero-typing is an expensive and time consuming process that requires implementing a battery of O and H antisera to detect 2541 different Salmonella enterica serovars. During this study a rapid multiplex Polymerase Chain Reaction (PCR) scheme was developed to screen for the prevalent Salmonella enterica serovar Enteritidis and Typhimurium. By analyzing the nucleotide sequences of th e genes for O-antigen biosynthesis including wba operon and the central variable regions of the H1 and H2 flagellin genes in Salmonella, designated PCR primers for two multiplex PCR reactions were used to detect and differentiate Salmonella serogroups A/D1, B, C1, C2, or E1; H1 antigen types i, g, m, r or z and H2 15 requiring a final confirmatory PCR test in the final serovar reporting of Salmonella enterica.

Demonstration of rapid multiplex PCR amplification involving 16 genetic loci

Current forensic DNA typing is conducted in approximately 8–10 h. Steps include DNA extraction, quantification, polymerase chain reaction (PCR) amplification of multiple short tandem repeat (STR) loci, capillary electrophoresis separation with fluorescence detection, data analysis and DNA profile interpretation. The PCR amplification portion of the workflow typically takes approximately 3 h with standard thermal cycling protocols. Here we demonstrate a rapid cycling protocol that amplifies 15 STR loci and the sex-typing marker amelogenin from the Identifiler STR typing kit in less than 36 min. This rapid protocol employs commercially available polymerases and the widely used GeneAmp 9700 thermal cycler. Complete concordance of STR allele calls (for 60 samples) between the rapid and standard thermal cycling protocols were observed although there was incomplete adenylation at several of the loci examined and some PCR artifacts were detected. Using less than 750 pg of template DNA and 28 cycles, STR peaks for all loci were above a 150 relative fluorescent unit (RFU) detection threshold with fully adequate inter-locus balance and heterozygote peak height ratios of greater than 0.84.

Simple Multiplex PCR for Rapid Diagnosis of Sex of Ducks and Duck Embryos

Haunshi, S., Saxena, S.C., Pattanayak, A., Bandyopadhaya, S., Tyagi, A.K. and Bujarbaruah, K.M. 2008. Simple multiplex PCR for rapid diagnosis of sex of ducks and duck embryos. J. Appl. Anim. Res., 33: 117–120. A simple and rapid multiplex polymerase chain reaction (PCR) was developed for quick diagnosis of sex of duck and duck embryos. W chromosome specific DNA sequence was selected and primers were designed to amplify 335 bp fragment from female sex while 16S ribosomal sequence was selected to design primers to amplify 468 bp PCR products both in male and female sex as an internal control. Nucleotide sequences of W chromosome specific DNA fragments of Khaki Campbell and Indian Runner breeds of duck were found to be identical both in size and sequences. The study concluded that the protocol was successful in precisely identifying the gender of ducklings belonging to both Indian Runner and Khaki Campbell breeds of ducks and duck embryos.

Simultaneous detection of three porcine viruses by multiplex PCR

Specific oligonucleotide primers were selected and combined in a multiplex arrangement, in order to detect simultaneously three economically important porcine viruses by polymerase chain reaction (PCR). The pathogen panel was comprised of viruses that cause reproductive failure in infected herds: Aujeszky's disease virus (ADV), porcine parvovirus (PPV) and porcine respiratory and reproductive syndrome virus (PRRSV). In order to reduce the time required for the detection of the pathogens, the assay was optimised to a RapidCycler PCR instrument. The multiplex PCR assay was shown to be specific, sensitive and rapid, because the results were read in less than 60 min after sample preparation. Due to its speed, efficiency and sensitivity, the described rapid multiplex PCR assay serves as a useful novel tool in the veterinary diagnostic laboratories for the quick and complex detection of these important porcine pathogens.

Multiplex Polymerase Chain Reaction for Microsatellite Analysis of Urine Sediment Cells: A Rapid and Inexpensive Method for Diagnosing and Monitoring Superficial Transitional Bladder Cell Carcinoma

Several urinary markers have been recently introduced in clinical practice for improving the noninvasive diagnosis of transitional cell carcinoma. Although microsatellite analysis must be considered the best method in terms of results, its cost and method time are unacceptable for daily use. We validated a more rapid and inexpensive method of determination using rapid DNA extraction and automatic multiplex polymerase chain reaction amplification. A total of 120 patients who presented consecutively to a urological office, including 73 with transitional cell carcinoma and 43 who served as controls, were selected for study. Microsatellite analysis was performed in the blood/urine pair using 3 multiplex polymerase chain reactions per patient. Urine sediment inflammatory cells were assessed by urine dipstick test. Ten microsatellite loci were investigated. Numerical data collected during electrophoresis of the amplified segment in an ABI Prism 310 Genetic Analyzer were used to calculate the cutoff for allelic imbalance. Method sensitivity, specificity, and positive and negative predictive values were calculated. A total of 66 patients had microsatellite analysis alterations in urine sediment, of whom 59 had transitional cell carcinoma, while 7 had other urological diseases. Test sensitivity and specificity were 80.8% and 85.1%, respectively. Statistical analysis did not indicate any significant influence of inflammatory status on microsatellite analysis diagnostic performance. In the control group the allelic imbalance on chromosome 9 was significantly lower than on other chromosomes (p = 0.0143). This could confirm that chromosome 9 has a specific role in transitional cell carcinoma. The multiplex microsatellite analysis method was low cost and not time-consuming. Multiplex microsatellite analysis is a noninvasive, rapid, inexpensive and reproducible method for screening for and monitoring superficial transitional cell carcinoma. It should be considered an alternative method to urinary cytology and it should also be considered in the presence of urine sediment inflammatory cells.

A multiplexed real-time PCR assay for rapid detection of Chlamydia trachomatis and identification of serovar L-2, the major cause of Lymphogranuloma venereum in New York

Lymphogranuloma venereum (LGV) is caused by a rare form of Chlamydia trachomatis that is difficult to diagnose, since culture is not readily available, and since other methods are not reliable or lack sensitivity. We report here a rapid, sensitive, and specific real-time multiplex polymerase chain reaction (PCR) assay capable of detecting C. trachomatis and identifying serovar L-2 in the same reaction, directly from rectal swabs. The analytical sensitivity of the assay was 25 genome copies for C. trachomatis, and 50 genome copies for L-2. The analytical specificity was 100%, as demonstrated with a diverse range of C. trachomatis serovars and other site-specific bacterial pathogens. With the use of a rapid DNA extraction method, a blinded validation of spiked rectal swabs correctly identified 30 samples containing C. trachomatis cells, L-2 DNA, or negative samples. The multiplexed PCR assay also identified serovar L-2 in 13 of 70 rectal swab samples taken from symptomatic patients. Twelve additional samples were positive for C. trachomatis only, and omp1 sequencing determined these samples as either serovar D, E, G, J, or K. This assay represents the first real-time PCR method capable of detecting C. trachomatis DNA, and of simultaneously identifying C. trachomatis infection as serovar L-2.