Multiplex real-time PCR assay combined with rolling circle amplification (MPRP) using universal primers for non-invasive detection of tumor-related mutations

The MPRP system for SNP discrimination was developed, which showed high specificity and sensitivity for multiplex detection of tumor-related mutations.

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Real-Time PCR Method for Quantification of Staphylococcus aureus in Milk

Journal of Food Protection ◽

10.4315/0362-028x-70.1.90 ◽

2007 ◽

Vol 70 (1) ◽

pp. 90-96 ◽

Cited By ~ 14

Author(s):

M. GOTO ◽

H. TAKAHASHI ◽

Y. SEGAWA ◽

H. HAYASHIDANI ◽

K. TAKATORI ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Real Time ◽

Real Time Pcr ◽

Pure Culture ◽

High Specificity ◽

Pcr Assay ◽

Milk Samples ◽

High Temperature Short Time ◽

Specificity And Sensitivity ◽

Pcr Method

A reproducible real-time PCR method that targets the putative transcriptional regulator gene of Staphylococcus aureus was developed to quantify this microorganism in milk samples. On the basis of partial sequences of this gene determined from S. aureus strains, we designed the specific primers and probe for use in a quantitative PCR assay. These specificities were confirmed with 25 strains of S. aureus and 35 strains of other bacteria. A real-time PCR assay with serial 10-fold dilutions of purified DNA and pure culture was conducted. It was possible to construct standard curves with a high correlation coefficient (r2 = 0.99) in the range of 50 ng to 50 fg for purified DNA and 107 to 101 CFU/ml for a pure culture. The constructed standard curve for milk samples was similar to that for the pure culture, and the quantification of S. aureus in the range of 107 to 101 CFU/ml was possible. Moreover, to determine how our real-time PCR method would perform under actual analytical conditions, we quantified the DNA from S. aureus after two types of heat treatments were used for the pasteurization of milk. The amount of DNA found was affected after heat treatment at 63°C for 30 min (low-temperature long-time method) but not at 72°C for 15 s (high-temperature short-time method). The results indicate that the real-time PCR method developed in this study is effective for monitoring S. aureus contamination in milk because of its high specificity and sensitivity.

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A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.00505-18 ◽

2018 ◽

Vol 56 (8) ◽

Cited By ~ 3

Author(s):

Nawal El Houmami ◽

Guillaume André Durand ◽

Janek Bzdrenga ◽

Anne Darmon ◽

Philippe Minodier ◽

...

Keyword(s):

Real Time ◽

Malate Dehydrogenase ◽

Real Time Pcr ◽

Detection Sensitivity ◽

Rt Pcr ◽

Pcr Assay ◽

Content Type ◽

Clinical Specimens ◽

Specificity And Sensitivity ◽

Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

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Preparation of Circular Templates by T4 RNA Ligase 2 for Rolling Circle Amplification of Target microRNAs with High Specificity and Sensitivity

Rolling Circle Amplification (RCA) ◽

10.1007/978-3-319-42226-8_3 ◽

2016 ◽

pp. 25-35

Author(s):

Yifu Guan ◽

Bin Zhao ◽

Guojie Zhao ◽

Chidong Xu ◽

Hong Shang

Keyword(s):

Rolling Circle Amplification ◽

High Specificity ◽

Rolling Circle ◽

Rna Ligase ◽

Specificity And Sensitivity ◽

T4 Rna Ligase

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Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Alternaria alternata

Journal of AOAC International ◽

10.5740/jaoacint.16-0196 ◽

2017 ◽

Vol 100 (1) ◽

pp. 99-103 ◽

Cited By ~ 3

Author(s):

Xinyue Zhang ◽

Guojie Xu ◽

Huaqi Tang ◽

Yanpeng Li ◽

Chunsheng Liu

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Alternaria Alternata ◽

Lamp Assay ◽

High Specificity ◽

Etiologic Agent ◽

Isothermal Amplification ◽

Pcr Assay ◽

The Real ◽

Loop Mediated Isothermal Amplification

Abstract Fungi of the Alternaria genus are associated with allergic diseases, with Alternaria alternata being one of the most prevalent species. A. alternata has been frequently reported as the etiologic agent of hypersensitivity pneumonitis, allergic rhinosinusitis, bronchial asthma,and other diseases. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay and a real-time PCR assay to detect low levels of A. alternata in herbal tea samples. The LAMP assay can detect as little as 3 pg/μL of A. alternata genomic DNA with high specificity. In addition, both the LAMP assay and the real-time PCR assay can be used for quantification of A. alternata. Although the newly developed LAMP assay is more rapid and specific in A. alternata identification, the real-time PCR assay is more precise in quantitation analysis.

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Quantitation of HBV covalently closed circular DNA in micro formalin fixed paraffin-embedded liver tissue using rolling circle amplification in combination with real-time PCR

Clinica Chimica Acta ◽

10.1016/j.cca.2011.06.031 ◽

2011 ◽

Vol 412 (21-22) ◽

pp. 1905-1911 ◽

Cited By ~ 27

Author(s):

Yanwei Zhong ◽

Jiaqi Han ◽

Zhengsheng Zou ◽

Shuhong Liu ◽

Bo Tang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Liver Tissue ◽

Rolling Circle Amplification ◽

Circular Dna ◽

Rolling Circle ◽

Covalently Closed Circular Dna ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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Evaluation of a stool antigen test using a mAb for native catalase for diagnosis of Helicobacter pylori infection in children and adults

Journal of Medical Microbiology ◽

10.1099/jmm.0.077370-0 ◽

2014 ◽

Vol 63 (12) ◽

pp. 1621-1625 ◽

Cited By ~ 10

Author(s):

Masumi Okuda ◽

Takako Osaki ◽

Shogo Kikuchi ◽

Junko Ueda ◽

Yingsong Lin ◽

...

Keyword(s):

Helicobacter Pylori ◽

Real Time ◽

Real Time Pcr ◽

High Specificity ◽

Antigen Test ◽

Stool Antigen Test ◽

Non Invasive ◽

Significant Difference ◽

Stool Antigen ◽

H Pylori

Non-invasive diagnosis of Helicobacter pylori infection is important not only for screening of infection but also for epidemiological studies. Stool antigen tests are non-invasive and are convenient to identify H. pylori infection, particularly in children. We evaluated the stool antigen test, which uses a mAb for native catalase of H. pylori developed in Japan. A total of 151 stool samples were collected from participants (52 children and 99 adults) of the Sasayama Cohort Study and stored between −30 and −80 °C. The stool antigen test used was Testmate pylori antigen (TPAg), and was performed according to the manufacturer’s instructions. Furthermore, we conducted a quantitative real-time PCR test and compared the PCR results with those of the TPAg test. When compared with the results in real-time PCR, the sensitivity of TPAg was 89.5 % overall, 82.7 % for children and 92.4 % for adults, and the specificity was 100 %. The accuracy was 93.4 % overall, 90.4 % for children and 94.9 % for adults, and there was no significant difference in the accuracy of TPAg between children and adults. Five of 28 children (18 %) and five of 38 adults (13 %) were PCR positive with negative TPAg results. Four of five children with positive PCR and negative TPAg results were given a 13C-urea breath test and all four children tested negative. No significant correlation was observed between the TPAg results and DNA numbers of H. pylori in faeces among children or adults. A stool antigen test (TPAg) using a mAb for native catalase is useful for diagnosis of H. pylori in children and adults. Additionally, this test has particularly high specificity.

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Development of a real-time PCR assay for the detection and identification of Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri

Journal of Medical Microbiology ◽

10.1099/jmm.0.47235-0 ◽

2007 ◽

Vol 56 (10) ◽

pp. 1346-1349 ◽

Cited By ~ 17

Author(s):

Tadayuki Iwase ◽

Keiko Seki ◽

Hitomi Shinji ◽

Yoshimitsu Mizunoe ◽

Shogo Masuda

Keyword(s):

Real Time ◽

Real Time Pcr ◽

High Specificity ◽

Pcr Assay ◽

Coagulase Negative Staphylococci ◽

Staphylococcus Warneri ◽

Staphylococcus Haemolyticus ◽

Staphylococcus Capitis ◽

Detection And Identification ◽

Species Specific

Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri are coagulase-negative staphylococci. Each species has different characteristics, and a difference in pathology is also seen in compromised hosts. Therefore, the development of a species-specific simple detection method for the identification of these staphylococci is important. Here, a species-specific real-time PCR assay is reported that targets the superoxide dismutase A-encoding gene of these bacteria. Primers were designed with a base that was non-complementary with regard to the other bacteria. This base was at the 3′ end of the primer (3′ mismatch primer) and conferred high specificity. These primers were then evaluated using real-time PCR. They reacted only with the target bacterium. In addition, stable quantitative reactions were observed when experiments were performed using genomic DNA extracted from varying numbers of staphylococci cells (101–107 cells). These results indicate that this method is useful for the identification and quantitative analysis of S. capitis, S. haemolyticus and S. warneri.

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