Library Quantification Using SYBR Green-Quantitative Polymerase Chain Reaction (qPCR)

2016 ◽  
Vol 2017 (6) ◽  
pp. pdb.prot094714 ◽  
Author(s):  
Elaine Mardis ◽  
W. Richard McCombie
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Sybr Green ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Novel Dual Labeled Fluorescence Probe Based Assay to Measure the Telomere Length

2018 ◽  
Author(s):  
Itty Sethi ◽  
Gh. Rasool Bhat ◽  
Rakesh Kumar ◽  
Ekta Rai ◽  
Swarkar Sharma
Keyword(s):  
Polymerase Chain Reaction ◽  
Telomere Length ◽  
Fluorescence Probe ◽  
Quantitative Polymerase Chain Reaction ◽  
Cost Effective ◽  
Sybr Green ◽  
Telomeric Dna ◽  
Chain Reaction ◽  
Specific Pcr ◽  
Polymerase Chain

ABSTRACTTelomeres are highly repetitive regions capping the chromosomes and composed of multiple units of hexa-nucleotides, TTAGGG, making their quantification difficult. Most of the methods developed to estimate telomeres are extensively cumbersome and expensive. The quantitative polymerase chain reaction (qPCR) based assay is relatively easy and cheaper method that applies SyBr Green dye chemistry to measure telomere length. As SyBr Green dye fluoresces after intercalation into the dsDNA, lack of differentiation between specific PCR target products and unspecific products is a limitation and it affects accuracy in quantitation of telomeres. To overcome the limitations of SyBr Green, we developed a dual labeled fluorescence probe based quantitative polymerase chain reaction (qPCR) to measure the telomere length. This robust, accurate and highly reproducible (R2=0.96) proprietary method (patent pending), yet cost effective and easy, utilizes a probe that targets specifically the telomeric DNA.

scholarly journals Evaluación de tres PCR cuantitativas para la detección de leptospiras patógenas en animales domésticos en Nicaragua

Biomédica ◽  
2020 ◽  
Vol 40 (4) ◽  
pp. 673-681
Author(s):  
Byron Flores ◽  
Nabil Halaihel ◽  
Tania Pérez-Sánchez ◽  
Jessica Sheleby-Elías ◽  
Brenda Mora ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Sybr Green ◽  
Chain Reaction ◽  
Polymerase Chain

Introducción. En Nicaragua es necesario estandarizar pruebas moleculares como la PCR en tiempo real (quantitative Polymerase Chain Reaction, qPCR) que mejoren el diagnóstico de leptospirosis en humanos y animales.Objetivo. Evaluar tres qPCR para la detección de leptospiras patógenas en animales domésticos de Nicaragua.Materiales y métodos. Se diseñaron cebadores para la amplificación del gen LipL32 en SYBR Green (SYBR Green-A) y TaqMan, y en otros descritos previamente (SYBR Green-B). Las secuencias de 12 cepas obtenidas de la base de datos del National Center for Biotechnology Information (NCBI) se alinearon para la búsqueda de sondas y cebadores. La sensibilidad analítica se determinó calculando el equivalente genómico detectable, se utilizaron 18 cepas de referencia para la sensibilidad diagnóstica y 28 controles negativos para la especificidad. Los métodos se aplicaron en 129 muestras de orina de animales domésticos.Resultados. En SYBR Green-A se obtuvo un límite de detección de cuatro equivalentes genómicos; en TaqMan, la sensibilidad fue del 94,4 % (IC95% 81,1-100,0). Con SYBR Green-A, se obtuvo una sensibilidad del 77,8 % (IC95% 55,8-99,8), en tanto que con SYBR Green-B fue del 61,1 % (IC95% 35,8-86,4). En las tres pruebas se logró una especificidad del 100 % (IC95% 98,2-100,0). El 26,4 % de las muestras de animales domésticos fueron positivas con SYBR Green-A y el 6,2 % con SYBR Green-B.Conclusiones. El SYBR Green-A presentó un límite de detección bajo, en tanto que las tres técnicas evaluadas mostraron alta especificidad, en tanto que la TaqMan tuvo la mayor sensibilidad.

scholarly journals APLIKASI KUANTIFIKASI KOI HERPESVIRUS : REAL TIME – QUANTITATIVE POLYMERASE CHAIN REACTION (RT-Q PCR) MENGGUNAKAN SYBR GREEN PADA IKAN MAS (Cyprinus carpio)

2017 ◽  
Vol 12 (1) ◽  
pp. 45
Author(s):  
Isti Koesharyani ◽  
Lila Gardenia ◽  
Tatik Mufidah ◽  
Ayi Santka
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Load ◽  
Real Time ◽  
Cyprinus Carpio ◽  
Quantitative Polymerase Chain Reaction ◽  
Sybr Green ◽  
Chain Reaction ◽  
Koi Herpesvirus ◽  
Ct Value ◽  
Polymerase Chain

Koi Herpes Virus (KHV) di Indonesia sejak tahun 2002 merupakan penyakit mematikan yang menyerang ikan koi Cyprinus carpio koi dan ikan mas Cyprinus carpio carpio, dan sampai saat ini, infeksi KHV dilaporkan sudah menyebar hampir di seluruh dunia. Untuk mengetahui adanya infeksi KHV perlu cara diagnosa yang sangat akurat/sensitif, sehingga keberadaan KHV dapat diketahui secara pasti dengan tingkat sensitivitas yang lebih baik pada ikan budidaya. Tujuan dari penelitian ini adalah untuk mengaplikasikan teknik deteksi dengan real time quantitative polymerase chain reaction (RT- qPCR/qPCR) guna mengetahui adanya infeksi KHV secara kuantitatif pada ikan mas dengan mengetahui kandungan virus (viral load). Sebanyak masing-masing 3 ekor sampel diperoleh dari sentra budidaya ikan mas di Cirata-Jawa Barat, Maninjau-Sumatera Barat, dan Banjarmasin-Kalimantan Selatan. Sampel-sampel tersebut selanjutnya dianalisa keberadaan KHV-nya dengan RT-qPCR menggunakan SYBR Green. Hasil pengujian menunjukkan bahwa jumlah tertinggi (viral load) diperoleh dari ikan mas asal Cirata-3 dengan nilai Threshold Cycle (Ct.) 18,24 atau setara dengan 3,4 x 107 kopi, dan terendah dari ikan mas asal Banjarmasin-3 dengan nilai Ct. 33,39 atau 1,8 x 102 kopi. Dua standar yang digunakan dalam pengujian ini berupa plasmid dengan jumlah kopi 2 x 104 (Ct 27,24) dan 2 x 103 (Ct 30,24) dan kontrol atau Non Template Control (NTC) adalah 3,1 x 10 atau dengan nilai Ct 35,65. Uji aplikasi deteksi KHV dengan metode RT-qPCR ini memberikan hasil yang lebih sensitif, di mana sampel yang tidak terdeteksi dengan metode PCR konvensional dapat dideteksi dan dihitung jumlah kopi DNA (DNA copy). Since 2002, Koi herpesvirus (KHV) in Indonesian has been a malignant diseases, now recognized as a worldwide cause of mortality among populations of koi Cyprinus carpio koi and common carp Cyprinus carpio carpio. To determine the presence of infection is required the KHV diagnosis method with highly accurate and sensitive, so that the existence KHV can be known exactly with high sensitivity level in fish farming.The objective of this study was to develop and evaluate the infection by Real Time Quantitative Polymerase Chain Reaction (RT- qPCR/qPCR). Sample were taken from Carp culture in West Java, West Sumatra, and South Kalimantan. The assay was done by SYBR Green RT-qPCR. The analysis result of KHV in carp revealed that the carp from Cirata-3 had the highest viral load with Ct. value 18.24 equal with 3.4 x 107 copies, and the lowest one was the carp from Banjarmasin-3 at Ct. value 33.39 (1.8 x 102 copies), while two standards plasmid and Non Template Control (NTC) had Ct value of 27.24 (2 x 104copies),30.24 (2 x 103copies), and 35.65 (3.1 x 10 copies), respectively. Application KHV test by q-PCR has more advantages and sensitive than that of conventional PCR, and it can be used to detect and calculate the copy number of DNA.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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