Rapid Real-time Polymerase Chain Reaction Research Articles

Development of a TaqMan Real-Time Polymerase Chain Reaction Assay for Detection of Burkholderia mallei

Burkholderia mallei is the causative agent of glanders, a highly contagious and reemerging zoonotic disease. B. mallei is closely related to Burkholderia pseudomallei, the etiologic agent of melioidosis. The genetic proximity between B. mallei and B. pseudomallei had always been a problem in development of specific molecular detection tests for B. mallei. In the present investigation, we describe a rapid, sensitive, and specific TaqMan real-time polymerase chain reaction (PCR) assay for detection of B. mallei. The TaqMan real-time PCR assay could detect as little as 1 picogram of B. mallei genomic DNA and 100 copies of the target DNA cloned in pGEM-T Easy vector. In spiked human blood, the assay could detect as few as 5.5 × 103 CFU/mL of B. mallei. The assay did not cross-react with other bacterial strains used in the study and was found specific for B. mallei. TaqMan real-time PCR assay can dramatically decrease the turnaround time for diagnostic results, and hence, appropriate action can be initiated at early stage to control and contain the B. mallei infection.

A one-step immune-chromatographic Helicobacter pylori stool antigen test for children was quick, consistent, reliable and specific.

This French study assessed a quick, noninvasive, immuno-chromatographic, Helicobacter pylori (H. pylori) stool antigen test for detecting infections in children. We enrolled 158 children, with a median age of 8.5 years (range eight months to 17 years), with digestive symptoms suggesting upper gastrointestinal tract disease. Upper digestive endoscopy was performed with gastric biopsy specimens for histology, a rapid urease test, culture test and quantitative real-time polymerase chain reaction. The H. pylori stool antigen test was performed twice for each child and the results were compared to the reference method. The reference methods showed that 23 (14.6%) of the 158 children tested were H. pylori positive. The H. pylori stool antigen test showed 91.3% sensitivity, with a 95% confidence interval (95% CI) of 86.9-95.6 and 97% specificity (95% CI 94.3-99.6), 30.84 positive likelihood ratio and 0.09 negative likelihood ratio. The test accuracy was 96.2% (95% CI 93.2-99.1). The two blinded independent observers produced identical H. pylori stool antigen test results and the Kappa coefficient for the H. pylori stool antigen test was one. The H. pylori stool antigen test was found to be a consistent, reliable, quick and specific test for detecting the H. pylori infection in children.

Comparison of nonstructural protein-1 antigen detection by rapid and enzyme-linked immunosorbent assay test and its correlation with polymerase chain reaction for early diagnosis of dengue.

INTRODUCTION:Early diagnosis of dengue is important for appropriate clinical management and vector control. Different serological tests based on the principle of immunochromatography and enzyme-linked immunosorbent assay (ELISA) are commonly used for detection of antigen and antibodies of dengue virus. The performance of these tests depends on the sensitivity and specificity. Hence, the study was undertaken to compare nonstructural protein-1 (NS1) antigen detection by rapid and ELISA with real-time polymerase chain reaction (RT-PCR) for diagnosis of dengue.MATERIALS AND METHODS:Prospective laboratory study was carried out on sera samples (n = 200) from clinically suspected cases of dengue. The sera samples were subjected for NS1 antigen detection test by rapid test, NS1 ELISA, and RT-PCR. The results of rapid and ELISA tests were compared with real Time PCR.RESULTS:The sensitivity, specificity, positive, and negative predictive value of rapid dengue NS1 antigen test were 81.5%, 66.7%, 78.2%, and 71.1%, respectively whereas that of NS1 ELISA were 89.9%, 100%, 100%, and 94%, respectively. Concordance of Rapid NS1 and NS1 ELISA with PCR was 75.5% and 94%.DISCUSSION AND CONCLUSION:NS1 antigen ELISA can be implemented in diagnostic laboratories for diagnosis of dengue in the acute phase of illness. The test also has great potential value for use in epidemic situations, as it could facilitate the early screening of patients and limit disease expansion.

On-site identification of meat species in processed foods by a rapid real-time polymerase chain reaction system

Correct labeling of foods is critical for consumers who wish to avoid a specific meat species for religious or cultural reasons. Therefore, gene-based point-of-care food analysis by real-time Polymerase Chain Reaction (PCR) is expected to contribute to the quality control in the food industry. In this study, we perform rapid identification of meat species by our portable rapid real-time PCR system, following a very simple DNA extraction method. Applying these techniques, we correctly identified beef, pork, chicken, rabbit, horse, and mutton in processed foods in 20min. Our system was sensitive enough to detect the interfusion of about 0.1% chicken egg-derived DNA in a processed food sample. Our rapid real-time PCR system is expected to contribute to the quality control in food industries because it can be applied for the identification of meat species, and future applications can expand its functionality to the detection of genetically modified organisms or mutations.

Asymptomatic and sub-microscopic malaria infection in Kayah State, eastern Myanmar

BackgroundMyanmar has the heaviest burden of malaria in the Greater Mekong Sub-region. Asymptomatic Plasmodium spp. infections are common in this region and may represent an important reservoir of transmission that must be targeted for malaria elimination.MethodsA mass blood survey was conducted among 485 individuals from six villages in Kayah State, an area of endemic but low transmission malaria in eastern Myanmar. Malaria infection was screened by rapid diagnostic test (RDT), light microscopy and real-time polymerase chain reaction (PCR), and its association with demographic factors was explored.ResultsThe prevalence of asymptomatic Plasmodium spp. infection was 2.3% (11/485) by real-time PCR. Plasmodium vivax accounted for 72.7% (8/11) and Plasmodium falciparum for 27.3% (3/11) of infections. Men were at greater risk of infection by Plasmodium spp. than women. Individuals who worked as farmers or wood and bamboo cutters had an increased risk of infection.ConclusionA combination of RDT, light microscopy and PCR diagnostics were used to identify asymptomatic malaria infection, providing additional information on asymptomatic cases in addition to the routine statistics on symptomatic cases, so as to determine the true burden of disease in the area. Such information and risk factors can improve malaria risk stratification and guide decision-makers towards better design and delivery of targeted interventions in small villages, representative of Kayah State.

Correlation between heteroplasmy level of the 5178C>A mutation in the ND2 gene of mitochondria DNA and essential hypertension in middle-aged and elderly adults

Objectives To explore the correlation between the heteroplasmy level of mt5178C>A mutation in ND2 gene of mitochondria DNA and essential hypertension(EH)in middle-aged and elderly adults. Methods EH patients and normotensive controls were recruited consecutively from 2014-2015 from general population.Demographics, clinical characteristics and blood leukocytes were collected.The mt5178C>A mutation heteroplasmy level was quantified by the rapid and sensitive real-time polymerase chain reaction(PCR)method for each participant. Results A total of 108 EH patients and 109 controls were recruited.The mt5178C>A mutation heteroplasmy level was(42±11%)in EH patients and(54±13)% in control subjects, with statistically significant difference between the two groups(P A heteroplasmy level exceeding 44% was associated with a decreased risk of EH(OR=0.18, 95%, CI: 0.10-0.31, P A heteroplasmy level was significantly negatively correlated with both systolic blood pressure(r=-0.38, P A heteroplasmy level(OR=0.82, 95%CI: 0.77-0.87, P A heteroplasmy level(OR=0.83, 95%CI: 0.78-0.89, P<0.01)was independent protective factor for EH, however, only total cholesterol(OR=2.17, 95%CI: 1.58-2.98, P=0.02)and low density lipoprotein cholesterol(OR=0.06, 95%CI: 0.01-0.83, P=0.04)were independent risk factors for EH, and the P at critical 0.05 value. Conclusions Mitochondrial ND2 gene 5178C>A mutation heteroplasmy level exerts protective role against EH in middle-aged and elderly adults in Chinese population. Key words: Hypertension; DNA, mitochondrial; Genetic heterogeneity

Development and validation of a real-time PCR assay for the detection of Toxoplasma gondii DNA in animal and meat samples.

We report a rapid and reliable method for the detection of Toxoplasma gondii in meat and animal tissues based on real-time polymerase chain reaction (PCR). Samples were collected from cattle, small ruminants, horses, and pigs raised or imported into Sicily, Italy. All DNA preparations were assayed by real-time PCR tests targeted to a 98-bp long fragment in the AF 529-bp repeat element and to the B1 gene using specific primers. Diagnostic sensitivity (100%), diagnostic specificity (100%), limit of detection (0.01 pg), efficiency (92-109%), and precision (mean coefficient of variation = 0.60%), repeatability (100%), reproducibility (100%), and robustness were evaluated using 240 DNA extracted samples (120 positives and 120 negative as per the OIE nested PCR method) from different matrices. Positive results were confirmed by the repetition of both real-time and nested PCR assays. Our study demonstrates the viability of a reliable, rapid, and specific real-time PCR on a large scale to monitor contamination with Toxoplasma cysts in meat and animal specimens. This validated method can be used for postmortem detection in domestic and wild animals and for food safety purposes.

Molecular-level evaluation of selected periodontal pathogens from subgingival regions in canines and humans with periodontal disease.

Dogs commonly serve as a model for various human conditions, including periodontal diseases. The aim of this study was to identify the anaerobic bacteria that colonize the subgingival areas in dogs and humans by using rapid real-time polymerase chain reaction (RT-PCR)-based tests and to compare the results obtained in each species. Bacterial microflora evaluations, both quantitative and qualitative, were performed by applying ready-made tests on twelve dogs and twelve humans. Five samples were collected from each subject's deepest gingival pockets and joined to form a collective sample. The results of the study revealed interspecies similarities in the prevalences of Porphyromonas (P.) gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum. Red complex bacteria comprised the largest portion of the studied bacterial complexes in all study groups, with P. gingivalis being the most commonly isolated bacterium. The results show similarities in the prevalence of bacterial microflora in dogs and humans. Microbiological analysis of gingival pockets by using rapid real-time PCR-based tests in clinical practice, both veterinary and human, can facilitate the choice of appropriate pharmacological treatment and can provide a basis for subsequent verification of the treatment's effectiveness.

Accuracy of a rapid real-time polymerase chain reaction assay for diagnosis of group B Streptococcus colonization in a cohort of HIV-infected pregnant women

Objective: There are limited data regarding Xpert performance to detect Group B Streptococcus (GBS) in HIV-infected pregnant women. We evaluated the accuracy of a rapid real-time polymerase chain reaction (PCR) test in a cohort of HIV-infected women.Methods: At 35–37 weeks of pregnancy, a pair of combined rectovaginal swabs were collected for two GBS assays in a cohort of sequentially included HIV-infected women in Rio de Janeiro: (1) culture; and (2) real-time PCR assay [GeneXpert GBS (Cepheid, Sunnyvale, CA)]. Using culture as the reference, sensitivity, specificity, positive and negative-likelihood ratios were estimated.Results: From June 2012 to February 2015, 337 pregnant women met inclusion criteria. One woman was later excluded, due to failure to obtain a result in the index test; 336 were included in the analyses. The GBS colonization rate was 19.04%. Sensitivity and specificity of the GeneXpert GBS assay were 85.94% (95% CI: 75.38–92.42) and 94.85% (95% CI: 91.55–96.91), respectively. Positive and negative predictive values were 79.71% (95% CI: 68.78–87.51) and 96.63% (95% CI: 93.72–98.22), respectively.Conclusions: GeneXpert GBS is an acceptable test for the identification of GBS colonization in HIV-infected pregnant women and represents a reasonable option to detect GBS colonization in settings where culture is not feasible.

Applications of a Rapid and Sensitive Dengue DUO Rapid Immunochromatographic Test Kit as a Diagnostic Strategy during a Dengue Type 2 Epidemic in an Urban City.

Dengue infection is a major health problem in tropical and subtropical countries. A prospective observational study in a university-affiliated hospital was conducted between August 2015 and September 2015. Patients who visited the emergency department (ED) with a presentation of any symptoms of dengue were eligible for the dengue non-structural protein 1 (NS1), IgM/IgG rapid immunochromatographic tests and real-time polymerase chain reaction (RT-PCR) to evaluate the performance of the rapid tests. Considering the RT-PCR as the gold standard for the dengue diagnosis, the ideal primary results of sensitivity (80–100%), specificity (60–84%), positive predicted value(75%-95%), and negative predicted value (70–100%) suggested that the NS1-based test with or without a combination of IgM and IgG tests have good diagnostic performances in detecting dengue infections, even in the afebrile or elderly populations.

Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology.

On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

Reactive case-detection of malaria in Pailin Province, Western Cambodia: lessons from a year-long evaluation in a pre-elimination setting.

BackgroundAs momentum towards malaria elimination grows, strategies are being developed for scale-up in elimination settings. One prominent strategy, reactive case detection (RACD), involves screening and treating individuals living in close proximity to passively detected, or “index” cases. This study aims to use RACD to quantify Plasmodium parasitaemia in households of index cases, and identify risk factors for infection; these data could inform reactive screening approaches and identify target risk groups.MethodsThis study was conducted in the Western Cambodian province of Pailin between May 2013 and March 2014 among 440 households. Index participants/index cases (n = 270) and surrounding households (n = 110) were screened for Plasmodium infection with rapid diagnostic tests (RDT), microscopy and real-time polymerase chain reaction (PCR). Participants were interviewed to identify risk factors. A comparison group of 60 randomly-selected households was also screened, to compare infection levels of RACD and non-RACD households. In order to identify potential risk factors that would inform screening approaches and identify risk groups, multivariate logistic regression models were applied.ResultsNine infections were identified in households of index cases (RACD approach) through RDT screening of 1898 individuals (seven Plasmodium vivax, two Plasmodium falciparum); seven were afebrile. Seventeen infections were identified through PCR screening of 1596 individuals (15 P. vivax, and 22 % P. falciparum/P. vivax mixed infections). In the control group, 25 P. falciparum infections were identified through PCR screening of 237 individuals, and no P. vivax was found. Plasmodium falciparum infection was associated with fever (p = 0.013), being a member of a control household (p ≤ 0.001), having a history of malaria infection (p = 0.041), and sleeping without a mosquito net (p = 0.011). Significant predictors of P. vivax infection, as diagnosed by PCR, were fever (p = 0.058, borderline significant) and history of malaria infection (p ≤ 0.001).ConclusionThis study found that RACD identified very few secondary infections when targeting index and neighbouring households for screening. The results suggest RACD is not appropriate, where exposure to malaria occurs away from the community, and there is a high level of treatment-seeking from the private sector. Piloting RACD in a range of transmission settings would help to identify the ideal environment for feasible and effective reactive screening methods.

A Rapid and Sensitive Detection of Aflatoxin-producing Fungus Using an Optimized Polymerase Chain Reaction (PCR).

Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxin-producing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was 65°C. The optimized template and primer concentration were 1.5 μL (50 ng/μL) and 3 μL (10 μM/μL) respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at 88.0°C, 87.5°C, 83.5°C, and 89.5°C respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.

On-chip quantitative detection of pathogen genes by autonomous microfluidic PCR platform.

Polymerase chain reaction (PCR)-based genetic testing has become a routine part of clinical diagnoses and food testing. In these fields, rapid, easy-to-use, and cost-efficient PCR chips are expected to be appeared for providing such testing on-site. In this study, a new autonomous disposable plastic microfluidic PCR chip was created, and was utilized for quantitative detection of pathogenic microorganisms. To control the capillary flow of the following solution in the PCR microchannel, a driving microchannel was newly designed behind the PCR microchannel. This allowed the effective PCR by simply dropping the PCR solution onto the inlet without any external pumps. In order to achieve disposability, injection-molded cyclo-olefin polymer (COP) of a cost-competitive plastic was used for the PCR chip. We discovered that coating the microchannel walls with non-ionic surfactant produced a suitable hydrophilic surface for driving the capillary flow through the 1250-mm long microchannel. As a result, quantitative real-time PCR with the lowest initial concentration of human, Escherichia coli (E. coli), and pathogenic E. coli O157 genomic DNA of 4, 0.0019, 0.031 pg/μl, respectively, was successfully achieved in less than 18 min. Our results indicate that the platform presented in this study provided a rapid, easy-to-use, and low-cost real-time PCR system that could be potentially used for on-site gene testing.

Evaluation of a Rapid One-step Real-time PCR Method as a High-throughput Screening for Quantification of Hepatitis B Virus DNA in a Resource-limited Setting.

Virological monitoring is the best predictor for the management of chronic hepatitis B virus (HBV) infections. Consequently, it is important to use the most efficient, rapid and cost-effective testing systems for HBV DNA quantification. The present study compared the performance characteristics of a one-step HBV polymerase chain reaction (PCR) vs the two-step HBV PCR method for quantification of HBV DNA from clinical samples. A total of 100 samples consisting of 85 randomly selected samples from patients with chronic hepatitis B (CHB) and 15 samples from apparently healthy individuals were enrolled in this study. Of the 85 CHB clinical samples tested, HBV DNA was detected from 81% samples by one-step PCR method with median HBV DNA viral load (VL) of 7.50 × 103 lU/ml. In contrast, 72% samples were detected by the two-step PCR system with median HBV DNA of 3.71 × 103 lU/ml. The one-step method showed strong linear correlation with two-step PCR method (r = 0.89; p < 0.0001). Both methods showed good agreement at Bland-Altman plot, with a mean difference of 0.61 log10 IU/ml and limits of agreement of -1.82 to 3.03 log10 IU/ml. The intra-assay and interassay coefficients of variation (CV%) of plasma samples (4-7 log10 IU/ml) for the one-step PCR method ranged between 0.33 to 0.59 and 0.28 to 0.48 respectively, thus demonstrating a high level of concordance between the two methods. Moreover, elimination of the DNA extraction step in the one-step PCR kit allowed time-efficient and significant labor and cost savings for the quantification of HBV DNA in a resource limited setting.How to cite this articleRashed-Ul Islam SM, Jahan M, Tabassum S. Evaluation of a Rapid One-step Real-time PCR Method as a High-throughput Screening for Quantification of Hepatitis B Virus DNA in a Resource-limited Setting. Euroasian J Hepato-Gastroenterol 2015;5(1):11-15.

Application of the SureTect Detection Methods for Listeria monocytogenes and Listeria spp. in Meat, Dairy, Fish, and Vegetable Products

The aim of this study was to evaluate the application of the Listeria monocytogenes and Listeria spp. SureTect detection methods for a rapid and sensitive detection in an ample range of food products: raw pork and poultry meat, Spanish chorizo, pate, smoked salmon, raw sheep milk cured cheese, and ready-to-eat lettuce salad. The combination of a 24-h enrichment in the 24 Listeria Enrichment Broth (LEB) coupled to a rapid bacterial DNA extraction and real-time polymerase chain reaction (RTi-PCR) using the specific SureTect methods detected down to 2–6 L. monocytogenes CFU per sample in less than 27 h on the food categories tested. Furthermore, the applicability of L. monocytogenes and Listeria spp. SureTect detection methods in real samples was assessed using 303 food samples, obtaining at least the same analytical performance as the international reference method ISO 11290-1.

Homogenous measurement during a circulation-water-based ultrahigh-speed polymerase chain reaction and melting curve analysis device

Polymerase chain reaction (PCR) is an essential technique for almost all life science fields that amplifies few target DNA copies. We developed a rapid real-time microdroplet PCR device by rapid switching of two types of circulation hot water, a µL-sized droplet, and a thin film aluminum chip. Using this device, rapid PCR amplification was observed successfully and accomplished within 3 min. By real-time PCR, many samples could be measured simultaneously. Moreover, melting curve analysis is essential for detecting differences of PCR amplicons in detail such as those related to nonspecific amplification, single nucleotide polymorphism, and genotyping methods. Thus, we developed a device that can be used for melting curve analysis using circulation water. From the obtained results, temperature homogeneity on the reaction plate was maintained during PCR and melting curve analysis using high-speed circulation water. The results indicate that this system can be applied to various life science fields.

Clinical applicability of rapid detection of SRY and DYS14 genes in patients with disorders of sex development using an indigenously developed 5′ exonuclease based assay

Life threatening conditions are associated with atypical genitalia in newborns. Analysis of genetic sex provides a clue to the underlying etiology in newborns with disorders of sex development (DSD) and can guide further endocrine investigations. Rapid diagnosis of genetic sex would be immensely useful in this situation. Traditionally used methods such as karyotype and fluorescence in situ hybridisation are time-consuming. To study the clinical applicability of an indigenously developed rapid real-time polymerase chain reaction (RT-PCR) assay for the sex determining region on the Y chromosome (SRY gene) and the DYS14 locus in newborns with DSD. Clinical examination, endocrinological tests, RT-PCR analysis of SRY and DYS14 and karyotype was performed in 15 newborns with DSD. RESULTS of PCR were available within 4 h. Based on this report, in SRY/DYS14 positive cases, further tests for assessment of testicular function were done. In SRY negative cases, tests for congenital adrenal hyperplasia were done. On comparing PCR results with other tests, the Y chromosome was present on karyotype and testicular tissue was detected by endocrinological and/or histological methods in all (8/15) SRY positive cases. The SRY and DYS14 negative cases (7/15) did not have Y chromosome in the karyotype. Congenital adrenal hyperplasia (CAH) was the most common diagnosis in this group. The indigenously developed PCR for dual Y chromosome markers is rapid and sensitive. Further endocrine evaluation of newborns with DSD can be based on these results. Information of genetic sex partly allays the psychosocial distress associated with the condition.

Molecular detection and corelation of Helicobacter pylori in dental plaque and gastric biopsies of dyspeptic patients

Helicobacter pylori is a microaerophilic organism, which colonizes in the gastric mucosa. Its role in etiology and development of acute and chronic gastritis and peptic ulcer diseases is scientifically proved. Oral cavity especially supragingival, subgingival plaque and so forth simulate the same microaerophilic environment favorable for the growth of this bacterium.Aim:Detection of H. pylori simultaneously in the oral cavity and gastric mucosa of patients suffering from gastric pathologies.Objectives:To detect H. pylori in the oral cavity and gastric mucosa using endoscopy, urease test and real-time polymerase chain reaction (PCR) (urease A gene). Determining its association and corelation with patient demographics, oral hygiene maintenance and periodontal disease status.Materials and Methods:Endoscopic examination, oral findings oral hygiene index-simplified (OHI-S) and community periodontal index and treatment needs (CPITN) indices were recorded. Antral biopsies and supragingival plaque samples were taken from 56 dyspeptic adult patients. The collected samples were subjected to histological examination, urease broth test and urease A gene amplification using real-time PCR.Result:H. pylori was detected in the supragingival plaque of individuals with H. pylori-induced gastric diseases using rapid urease test and real-time PCR analysis. Occurrence of same strain of H. pylori simultaneously in plaque and gastric mucosa was observed. Positive correlation was obtained between the collected indices and quantity of H. pylori colonization.

Evaluation of real-time PCR for quantitative detection of Escherichia coli in beach water

The current investigation evaluated the use of real-time polymerase chain reaction (PCR) for quantitative detection of Escherichia coli in marine beach water. Densities of E. coli in 263 beach water samples collected from 13 bathing beaches in Hong Kong between November 2008 and December 2009 were determined using both real-time PCR and culture-based methods. Regression analysis showed that these two methods had a significant positive linear relationship with a correlation coefficient (r) of 0.64. Serial dilution of spiked samples indicated that the real-time PCR had a limit of quantification of 25 E. coli colonies in 100 mL water sample. This study showed that the rapid real-time PCR has potential to complement the traditional culture method of assessing fecal pollution in marine beach water.