Polymerase chain reaction (PCR) v2

Salmonella typhi is bacteria that cause typhoid disease in humans. In Indonesia, the morbidity number of typhoid disease tends to be increase. Thus, it has been requiring the alternative for handling or preventing that disease. Recently, the detection method commonly uses for S. typhi detection is Serological test. The weakness of this method is often producing less accurate and not specific detection. The previous research was successfully discovered S. typhi gene that codes protein which is contributed at adherents or colonization those bacteria in epithelial human cell. That result was base to develop detection on S. typhi method by Polymerase chain reaction (PCR). The aim of this research is developing a specific and accurate detection method for S. typhi bacteria by PCR. The research result is performed successfully to amplify the fimbrial-C S. typhi gene using pairs of primer FW-INT 2- REV-1A NEW which was designed and synthesized in previous step. That success showed by the finding of the DNA fragment of 0.2 kilobase (kb) proffers to size of DNA fragment which is hopefully in using S. typhi genome as a template. Specificity and sensitivity test for those primers are still conducting to reproducibility results. Base on the results can be concluded that the research have successfully conducted in developing S. typhi detection method using pairs of S. typhi fimbrial-C primer. Hopefully, the studied of developing detection methods was conducted better compare with former detection methods.Keywords: S. typhi detection method, fim-C S. typhi gene, PCRAbstrakSalmonella typhi merupakan bakteri penyebab penyakit tifus pada manusia. Di Indonesia, angka morbiditas penderita penyakit typhus cenderung meningkat, sehingga diperlukan suatu alternatif untuk penanganan atau pencegahan penyakit tersebut. Sampai saat ini metode deteksi S. typhi yang banyak digunakan adalah uji serologi. Kelemahan metode ini adalah sering menghasilkan deteksi yang kurang akurat dan tidak spesifik. Pada penelitian yang dilakukan sebelumnya, telah berhasil ditemukan gen fimbrial-C S. typhi pengkode protein yang berperan dalam penempelan S. typhi pada usus manusia, hasil ini dijadikan landasan untuk pengembangan metode deteksi menggunakan teknik PCR. Tujuan penelitian ini mengembangkan metode deteksi yang akurat dan spesifik untuk bakteri penyebab penyakit typhus pada manusia. Hasil penelitian menunjukkan bahwa telah berhasil dilakukan amplifikasi gen fimbrial-C S. typhi menggunakan pasangan primer hasil perancangan yaitu FW-INT 2- REV-1A NEW. Keberhasilan tersebut ditunjukkan dengan diperolehnya pita DNA berukuran 0.2 kilo basa (kb) sesuai dengan ukuran pita DNA yang diharapkan dengan menggunakan template DNA genom bakteri S. typhi. Uji sensitivitas dan spesifisitas terhadap primer hasil rancangan sedang di kaji lebih lanjut untuk memperoleh reprodusibiltas hasil pengujian. Berdasarkan hasil yang diperoleh dapat disimpulkan bahwa telah berhasil dilakukan pengembangan metode deteksi S. typhi menggunakan pasangan primer fimbrial-C S. typhi. Pengkajian pengembangan metode deteksi yang dihasilkan ini diharapkan dapat lebih baik dibanding beberapa metode deteksi yang sudah ada.Kata Kunci: Metode Deteksi Bakteri typhus, fim-C S. typhi, PCR

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Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

Canadian Journal of Microbiology ◽

10.1139/w05-140 ◽

2006 ◽

Vol 52 (5) ◽

pp. 451-461 ◽

Cited By ~ 8

Author(s):

S S Hynes ◽

O Chaudhry ◽

M A Providenti ◽

M L Smith

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Genetically Modified Organisms ◽

Quantitative Polymerase Chain Reaction ◽

Pcr Primers ◽

Chain Reaction ◽

Environmental Persistence ◽

Fungal Strains ◽

Target Dna ◽

Polymerase Chain

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (~14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.Key words: Canada domestic substances list (DSL), Canadian Environmental Protection Act (CEPA), genetically modified organisms (GMO), quantitative polymerase chain reaction (qPCR).

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Evaluation of Resistance to Rhabdocline Needlecast in Douglas Fir Variety Shuswap, with Quantitative Polymerase Chain Reaction

Phytopathology ◽

10.1094/phyto-100-4-0337 ◽

2010 ◽

Vol 100 (4) ◽

pp. 337-344 ◽

Cited By ~ 8

Author(s):

M. Catal ◽

G. C. Adams ◽

D. W. Fulbright

Keyword(s):

Polymerase Chain Reaction ◽

Quantitative Polymerase Chain Reaction ◽

Douglas Fir ◽

Chain Reaction ◽

Disease Symptoms ◽

Seed Sources ◽

Release Times ◽

Spore Release ◽

Target Dna ◽

Polymerase Chain

A quantitative polymerase chain reaction assay was developed that could detect DNA of Rhabdocline pseudotsugae and R. oblonga among DNA of Douglas fir needles to a limit as low as three copies of target DNA. Differential infection rates of two varieties (seed sources) of Douglas fir interplanted in a field were studied in relation to staggered bud breaks. Infection of Douglas fir var. San Isabel corresponded to ascospore release times for Rhabdocline spp., whereas infection of var. Shuswap Lake did not occur throughout the spore release period during 2 years of study, despite abundant inoculum and adequate moisture during bud break. Rhabdocline spp. DNA was never detected in Shuswap Lake and disease symptoms were not observed in any year. We provide evidence that Shuswap Lake is resistant and probably immune to Rhabdocline spp. infection and Rhabdocline needlecast under Michigan conditions.

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Performance of a Commercial Polymerase Chain Reaction Test for EndocervicalChlamydia trachomatisInfection in a University Hospital Population

Infectious Diseases in Obstetrics and Gynecology ◽

10.1155/s1064744998000453 ◽

1998 ◽

Vol 6 (5) ◽

pp. 224-229

Author(s):

C. H. Livengood III ◽

K. A. Boggess ◽

J. W. Wrenn ◽

A. P. Murtha

Keyword(s):

Polymerase Chain Reaction ◽

Polymerase Chain Reaction Test ◽

Laboratory Evaluation ◽

Clinical Samples ◽

Chain Reaction ◽

Predictive Values ◽

Target Dna ◽

Wide Range ◽

Polymerase Chain ◽

Pcr Test

Objectives:To examine the accuracy of a commercial polymerase chain reaction (PCR) test (Amplicor CTR, Roche Diagnostic Systems, Branchburg NJ) for identification of endocervical chlamydial infections through both laboratory evaluation and among a diverse teaching hospital patient population.Methods:Testing of reliable threshold inocula and reproducibility were carried out using laboratory stock organisms. Paired endocervical samples from patients with a wide range of indications were tested by PCR and an established culture procedure, and discrepant pairs were further analyzed to determine true results.Results:Laboratory evaluation suggested that one copy of target DNA from a viable organism consistently yielded a positive result, and test reproducibility was very good, with an overall coefficient of variation of 15%. Compared to true results in 1,588 paired clinical samples from 1,489 women with a 10% prevalence of infection, the PCR test and culture yielded respective sensitivities of 87.4% and 78.0%, and negative predictive values of 98.6% and 97.6%. Specificity and positive predictive value for both tests were 100%. Cost per specimen was nearly identical at $18.84 and $18.88 respectively. Polymerase inhibitors and organisms lacking target DNA were not found in false-negative PCR samples.Conclusion:This commercial PCR test is accurate, cost-competitive, and much faster than culture for diagnosis of endocervical chlamydia infections in our population of intermediate prevalence of chlamydial infection.

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Plasma vs. serum in circulating tumor DNA measurement: characterization by DNA fragment sizing and digital droplet polymerase chain reaction

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2019-0896 ◽

2020 ◽

Vol 58 (4) ◽

pp. 527-532 ◽

Cited By ~ 3

Author(s):

Jee-Soo Lee ◽

Miyoung Kim ◽

Moon-Woo Seong ◽

Han-Sung Kim ◽

Young Kyung Lee ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Circulating Tumor Dna ◽

Analytical Sensitivity ◽

Dna Fragments ◽

Chain Reaction ◽

Specimen Type ◽

Dna Fragment ◽

Polymerase Chain ◽

Ctdna Analysis ◽

Tumor Dna

AbstractBackgroundChoosing the specimen type is the first step of the pre-analytical process. Previous reports suggested plasma as the optimal specimen for circulating tumor DNA (ctDNA) analysis. However, head-to-head comparisons between plasma and serum using platforms with high analytical sensitivity, such as droplet digital polymerase chain reaction (ddPCR), are limited, and several recent studies have supported the clinical utility of serum-derived ctDNA. This study aimed to compare the DNA profiles isolated from plasma and serum, characterize the effects of the differences between specimens on ctDNA measurement, and determine the major contributors to these differences.MethodsWe isolated cell-free DNA (cfDNA) from 119 matched plasma/serum samples from cancer patients and analyzed the cfDNA profiles by DNA fragment sizing. We then assessed KRAS mutations in ctDNA from matched plasma/serum using ddPCR.ResultsThe amount of large DNA fragments was increased in serum, whereas that of cfDNA fragments (<800 bp) was similar in both specimens. ctDNA was less frequently detected in serum, and the KRAS-mutated fraction in serum was significantly lower than that in plasma. The differences in ctDNA fractions between the two specimen types correlated well with the amount of large DNA fragments and white blood cell and neutrophil counts.ConclusionsOur results provided detailed insights into the differences between plasma and serum using DNA fragment sizing and ddPCR, potentially contributing to ctDNA analysis standardization. Our study also suggested that using plasma minimizes the dilution of tumor-derived DNA and optimizes the sensitivity of ctDNA analysis. So, plasma should be the preferred specimen type.

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