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

2020 ◽  
Vol 58 (4) ◽  
pp. 527-532 ◽  
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.

scholarly journals Analytic and Clinical Validation of an Ultrasensitive, Quantitative Polymerase Chain Reaction Assay for EGFR Mutation Analysis With Circulating Tumor DNA

2017 ◽  
Vol 141 (7) ◽  
pp. 978-984 ◽  
Author(s):  
Xiaowei Wang ◽  
Yunhua Gao ◽  
Bei Wang ◽  
Zhenrong Zhang ◽  
Chaoyang Liang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Mutation Analysis ◽  
Polymerase Chain Reaction Assay ◽  
Egfr Mutation ◽  
Quantitative Polymerase Chain Reaction ◽  
Circulating Tumor Dna ◽  
Egfr Mutations ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tumor Dna

Context.— The mutation analysis of epidermal growth factor receptor (EGFR) has become a common test to guide therapeutic decision making for lung cancer. Molecular testing with circulating tumor DNA in plasma allows diagnosis of mutations when tumor tissue is not available as well as monitoring treatment response with repeat biopsies. Objectives.— To develop a timely and cost-effective assay that can accurately detect EGFR mutations in circulating tumor DNA and to evaluate the analytic and clinical performance of the assay. Design.— Analytic assessment was conducted with a set of reference materials carrying classic EGFR mutations. A recently developed Poisson distribution–based approach was employed to understand the assay sensitivity. Clinical evaluation was performed with 224 pairs of plasma and matched tissues from patients with stage I to IV disease. EGFR mutation rates of 390 consecutive plasma samples processed in the central service laboratory were compared with previously reported prevalence in an Asian population. Results.— Our results suggested that limit of detection for the EGFR quantitative polymerase chain reaction assay was 10 mutation copies, and the lowest detectable copy numbers could be extended to a single-digit level. The clinical sensitivity was 53.3% for all stages combined and 81.4% for late stages, with a high specificity of 100%. Clinical observations showed an overall positive finding rate of 32.5% and 41.4% for stage IV disease, which is consistent with previously reported EGFR mutation prevalence in an Asian population. Conclusions.— Our results supported the clinical utility of the ultrasensitive, quantitative polymerase chain reaction assay for EGFR mutation analysis with circulating tumor DNA.

scholarly journals DETEKSI BAKTERI PENYEBAB PENYAKIT TYPHUS PADA MANUSIA DENGAN POLYMERASE CHAIN REACTION

2011 ◽  
Vol 1 (1) ◽  
pp. 45
Author(s):  
Muktiningsih Nurjayadi ◽  
Fera Kurnia Dewi ◽  
Dahlia Dahlia ◽  
S, Restu.N S ◽  
Fitri W
Keyword(s):  
Polymerase Chain Reaction ◽  
Detection Method ◽  
Serological Test ◽  
Research Result ◽  
Salmonella Typhi ◽  
Detection Methods ◽  
Chain Reaction ◽  
Specificity And Sensitivity ◽  
Dna Fragment ◽  
Polymerase Chain

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

scholarly journals Field evaluation of a quantitative polymerase chain reaction assay for Mycoplasma hyorhinis

2014 ◽  
Vol 26 (6) ◽  
pp. 755-760 ◽  
Author(s):  
Maria J. Clavijo ◽  
Simone Oliveira ◽  
Jeffrey Zimmerman ◽  
Aaron Rendahl ◽  
Albert Rovira
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
Systemic Disease ◽  
Analytical Sensitivity ◽  
Upper Respiratory Tract ◽  
Chain Reaction ◽  
Mycoplasma Hyorhinis ◽  
Field Samples ◽  
Polymerase Chain

Mycoplasma hyorhinis has emerged as an important cause of systemic disease in nursery pigs. However, this bacterium can also be found in the upper respiratory tract of healthy swine. The current study describes the development of a quantitative polymerase chain reaction assay for the detection of M. hyorhinis and the evaluation of the assay in both disease diagnosis and disease surveillance using a large number of field samples. The analytical sensitivity was estimated to be 12 genome equivalents/μl. The assay was highly specific, detecting all 25 M. hyorhinis isolates tested and none of the 19 nontarget species tested. Assay repeatability was evaluated by testing different matrices spiked with known amounts of M. hyorhinis. Overall, assessment of the repeatability of the assay showed suitable precision within and between runs for all matrices. The coefficient of variation ranged from 10% to 24%. Mycoplasma hyorhinis DNA was detected in 48% of samples (pericardium, pleura, joints, nasal cavity, and lungs) from pigs with systemic disease. Mycoplasma hyorhinis was detected in nasal (92%) and oropharyngeal swabs (66%), as well as in oral fluids (100%). Potential uses of this tool involve the characterization of the prevalence of this pathogen in swine herds as well as bacterial quantification to evaluate intervention efficacy.

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