Comparison of Real Time PCR Cycle Threshold Values in Symptomatic and Asymptomatic COVID-19 Patients

2021 ◽  
Vol 55 (3) ◽  
pp. 435-444
Author(s):  
Harun Gülbudak ◽  
Şinasi Karvar ◽  
Gizem Soydan ◽  
Seda Tezcan Ülger ◽  
Özlem Kandemir ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Threshold Values ◽  
Cycle Threshold

scholarly journals Assessment of Commercial DNA Cleanup Kits for Elimination of Real-Time PCR Inhibitors in the Detection of Cyclospora cayetanensis in Cilantro

2020 ◽  
Vol 83 (11) ◽  
pp. 1863-1870
Author(s):  
ANGELA ASSURIAN ◽  
HELEN MURPHY ◽  
ALICIA SHIPLEY ◽  
HEDIYE NESE CINAR ◽  
ALEXANDRE DA SILVA ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Threshold Values ◽  
Cyclospora Cayetanensis ◽  
Cycle Threshold ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Inhibitor ◽  
Zymo Research

ABSTRACT Inhibited reactions have occasionally been observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using quantitative real-time PCR (qPCR). Partial or total inhibition of PCR reactions, including qPCR, can occur, leading to decreased sensitivity or false-negative results. If inhibition occurs, this implies the need for additional purification or cleanup treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA cleanup kits (QIAquick purification kit from Qiagen [kit 1], OneStep PCR inhibitor removal by Zymo Research [kit 2], NucleoSpin genomic DNA cleanup XS from Macherey-Nagel [kit 3], DNA IQ system by Promega [kit 4], and DNeasy PowerPlant pro kit from Qiagen [5]) to minimize qPCR inhibition using the U.S. Food and Drug Administration–validated Bacteriological Analytical Manual (BAM) Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA cleanup kits evaluated was able to reduce the qPCR internal amplification control cycle threshold values to those considered to be normal for noninhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both a high oocyst level (200 oocysts) and a low oocyst level (10 oocysts). Of the five kits compared, kits 1, 2, and 3 did not show significant differences in the detection of C. cayetanensis, while significantly higher cycle threshold values, indicating lower recovery of the target DNA, were observed from kits 4 and/or 5 in samples seeded with 200 and 10 oocysts (P < 0.05). This comparative study provides recommendations on the use of commercial cleanup kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using the BAM Chapter 19b method. HIGHLIGHTS

scholarly journals Preanalytical Issues and Cycle Threshold Values in SARS-CoV-2 Real-Time RT-PCR Testing: Should Test Results Include These?

ACS Omega ◽  
2021 ◽  
Author(s):  
Ilka Engelmann ◽  
Enagnon Kazali Alidjinou ◽  
Judith Ogiez ◽  
Quentin Pagneux ◽  
Sana Miloudi ◽  
...  
Keyword(s):  
Real Time ◽  
Test Results ◽  
Threshold Values ◽  
Rt Pcr ◽  
Cycle Threshold

Detection of Rendered Meat and Bone Meals by PCR Is Dependent on Animal Species of Origin and DNA Extraction Method

2010 ◽  
Vol 73 (6) ◽  
pp. 1090-1096 ◽  
Author(s):  
MICHAEL J. MYERS ◽  
DOROTHY E. FARRELL ◽  
CHRISTINE M. DEAVER ◽  
JACQULINE MASON ◽  
HEIDI L. SWAIM ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Animal Species ◽  
Animal Feed ◽  
Functional Assay ◽  
Threshold Values ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Pcr Method

The capability of eight commercially available DNA extraction kits to extract bovine DNA originating in meat and bone meal from fortified feed was evaluated. Four different batches of bovine meat and bone meal (BMBM) were used for DNA extraction with the eight commercial DNA extraction kits. Within each kit, there were minimal differences in the batch-to-batch amounts of extracted DNA. There were differences between the kits in the amounts of DNA that could be extracted from the same amount of starting BMBM. These differences did not translate into differences in the amount of amplifiable DNA from BMBM-fortified dairy feed. Using a validated real-time PCR method, the kit yielding the highest amount extractable DNA was completely unable to yield a positive PCR result; one other kit was also unable to produce a positive PCR result from DNA extracted from BMBM-fortified feed. There was a complete lack of a correlation between the amount of bovine DNA isolated from BMBM by a given extraction kit compared with the relative amounts of DNA isolated from fortified animal feed as evidenced by the cycle threshold values generated using the real-time PCR method. These results demonstrate that extraction of DNA from processed animal protein is different for pure ingredients and fortified animal feeds. These results indicate that a method specifically developed using just animal-derived meat and bone meal may not yield a functional assay when used to detect animal tissues in complete animal feed.

scholarly journals Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR

2015 ◽  
Vol 142 (5) ◽  
pp. 555 ◽  
Author(s):  
BVishnu Bhat ◽  
DBenet Bosco Dhas ◽  
AHiasindh Ashmi ◽  
SubashChandra Parija ◽  
N Banupriya
Keyword(s):  
Real Time ◽  
Melting Temperature ◽  
Real Time Pcr ◽  
Low Cost ◽  
Snp Genotyping ◽  
Cycle Threshold ◽  
Allele Specific

Factors associated with real-time RT-PCR cycle threshold values among medically attended influenza episodes

2015 ◽  
Vol 88 (4) ◽  
pp. 719-723 ◽  
Author(s):  
Sarah Spencer ◽  
Jessie Chung ◽  
Mark Thompson ◽  
Pedro A. Piedra ◽  
Alan Jewell ◽  
...  
Keyword(s):  
Real Time ◽  
Threshold Values ◽  
Rt Pcr ◽  
Factors Associated ◽  
Cycle Threshold

scholarly journals Incorporating temporal distribution of population-level viral load enables real-time estimation of COVID-19 transmissibility

2021 ◽  
Author(s):  
Yun Lin ◽  
Bingyi Yang ◽  
Sarah Cobey ◽  
Eric Lau ◽  
Dillon Adam ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Temporal Distribution ◽  
Time Estimation ◽  
Population Level ◽  
Reproductive Number ◽  
Threshold Values ◽  
Illness Onset ◽  
Cycle Threshold ◽  
Viral Loads

Abstract Many locations around the world have used real-time estimates of the time-varying effective reproductive number (\({R}_{t}\)) of COVID-19 to provide evidence of transmission intensity to inform control strategies. Estimates of \({R}_{t}\) are typically based on statistical models applied to case counts and typically suffer lags of more than a week because of the incubation period and reporting delays. Noting that viral loads tend to decline over time since illness onset, analysis of the distribution of viral loads among confirmed cases can provide insights into epidemic trajectory. Here, we analyzed viral load data on confirmed cases during two local epidemics in Hong Kong, identifying a strong correlation between temporal changes in the distribution of viral loads (measured by cycle threshold values) and estimates of \({R}_{t}\) based on case counts. We demonstrate that cycle threshold values could be used to improve real-time \({R}_{t}\) estimation, enabling more timely tracking of epidemic dynamics.

scholarly journals Identifikasi Penambahan Daging Babi pada Pangan Berbahan Dasar Daging Sapi Menggunakan ELISA dan qPCR

2019 ◽  
Vol 7 (2) ◽  
pp. 17-25
Author(s):  
Diyan Cahyaningsari ◽  
Hadri Latif ◽  
Etih Sudarnika
Keyword(s):  
Real Time ◽  
Optical Density ◽  
Immunosorbent Assay ◽  
Real Time Pcr ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cycle Threshold

Penelitian ini bertujuan untuk mengidentifikasi dan menganalisis penambahan daging babi ternak dan babi hutan baik yang mentah (raw) maupun yang diolah (cooked) di dalam pangan asal hewan berbahan dasar daging sapi menggunakan metode uji enzyme-linked immunosorbent assay (ELISA)  dan real-time PCR (qPCR). Sebanyak 40 sampel yang terdiri dari 20 daging babi ternak dan 20 daging babi hutan dihomogenisasi dengan daging sapi dalam bentuk mentah maupun olahan (bakso). Konsentrasi setiap daging babi ternak dan babi hutan dalam bentuk mentah dan olahan bakso pada campuran daging sapi antara lain 2%, 1%, 0.5%, 0.25%, dan 0.125%. Hasil uji ELISA pada penelitian ini menunjukkan bahwa uji ini mampu mengidentifikasi adanya penambahan daging babi ternak dan babi hutan dalam pangan asal hewan baik dalam bentuk mentah maupun olahan bakso hingga konsentrasi 0.25%. Hasil uji t terhadap nilai optical density (OD) uji ELISA menunjukkan bahwa tidak terdapat perbedaan dalam mendeteksi spesies babi ternak dengan babi hutan (p≥0.05), tetapi terdapat perbedaan pada sampel bentuk mentah dengan bakso (p<0.05). Hasil uji qPCR mampu mengidentifikasi adanya penambahan daging babi ternak dan babi hutan dalam pangan asal hewan baik dalam bentuk mentah maupun olahan bakso hingga konsentrasi 0.125%. Hasil uji t  terhadap nilai cycle threshold (Ct) uji qPCR menunjukkan bahwa tidak terdapat perbedaan dalam mendeteksi spesies babi ternak dengan babi hutan (p≥0.05), tetapi terdapat perbedaan pada sampel bentuk mentah dengan bakso (p<0.05). Metode ELISA dan qPCR dapat dijadikan sebagai metode pengujian untuk mengidentifikasi pencampuran spesies yang tidak dikehendaki, khususnya daging babi pada pangan asal hewan berbahan dasar daging sapi yang beredar di masyarakat.

OPTIMASI VOLUME TEMPLAT DNA DAN JUMLAH SIKLUS AMPLIFIKASI UNTUK DETEKSI Wuchereria bancrofti METODE REAL-TIME PCR

2019 ◽  
Vol 11 (2) ◽  
pp. 160
Author(s):  
Anisa Safanah ◽  
Ai Djuminar ◽  
Fusvita Merdekawati ◽  
Entuy Kurniawan ◽  
Ernawati Ernawati
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gold Standard ◽  
Wuchereria Bancrofti ◽  
Cycle Threshold

Filariasis yang disebabkan oleh Wuchereria bancrofti masih menjadi masalah kesehatan di Indonesia. Pemeriksaan mikroskopis darah sebagai gold standard memiliki beberapa kelemahan, sehingga diagnosis klinis berbasis Biologi Molekuler mulai dikembangkan, khususnya metode Real-Time PCR. Pada penelitian ini untuk  mencapai hasil terbaik dilakukan optimasi terhadap volume templat DNA dan jumlah siklus amplifikasi. Tujuan penelitian ini adalah mengetahui berapa volume templat DNA dan jumlah siklus amplifikasi yang optimum untuk deteksi Wuchereria bancrofti dengan menggunakan metode Real-Time PCR. Jenis penelitian yang digunakan adalah eksperimen semu. Desain penelitian yang dilakukan yaitu dibuat matriks terhadap volume templat DNA dan jumlah siklus amplifikasi yaitu 2;30, 3;30, 4;30, 2;35, 3;35, 4;35, 2;40, 3;40, 4;40, kemudian dilakukan pemeriksaan dari produk templat DNA Wuchereria bancrofti. Analisis data menggunakan grafik hasil amplifikasi berupa nilai Ct (cycle threshold). Dari hasil penelitian menunjukkan kondisi optimal untuk volume templat DNA adalah 2µL, sedangkan untuk jumlah siklus amplifikasi adalah 35 siklus.

Assessment of real-time PCR cycle threshold values in Microsporum canis culture-positive and culture-negative cats in an animal shelter: a field study

2017 ◽  
Vol 20 (2) ◽  
pp. 108-113 ◽  
Author(s):  
Linda S Jacobson ◽  
Lauren McIntyre ◽  
Jenny Mykusz
Keyword(s):  
Field Study ◽  
Real Time ◽  
Real Time Pcr ◽  
Fungal Culture ◽  
Time Points ◽  
Cycle Threshold ◽  
Ct Value ◽  
Two Samples ◽  
Culture Positive ◽  
Culture Negative

Objectives Real-time PCR provides quantitative information, recorded as the cycle threshold (Ct) value, about the number of organisms detected in a diagnostic sample. The Ct value correlates with the number of copies of the target organism in an inversely proportional and exponential relationship. The aim of the study was to determine whether Ct values could be used to distinguish between culture-positive and culture-negative samples. Methods This was a retrospective analysis of Ct values from dermatophyte PCR results in cats with suspicious skin lesions or suspected exposure to dermatophytosis. Results One hundred and thirty-two samples were included. Using culture as the gold standard, 28 were true positives, 12 were false positives and 92 were true negatives. The area under the curve for the pretreatment time point was 96.8% (95% confidence interval [CI] 94.2–99.5) compared with 74.3% (95% CI 52.6–96.0) for pooled data during treatment. Before treatment, a Ct cut-off of <35.7 (approximate DNA count 300) provided a sensitivity of 92.3% and specificity of 95.2%. There was no reliable cut-off Ct value between culture-positive and culture-negative samples during treatment. Ct values prior to treatment differed significantly between the true-positive and false-positive groups ( P = 0.0056). There was a significant difference between the pretreatment and first and second negative culture time points ( P = 0.0002 and P <0.0001, respectively). However, there was substantial overlap between Ct values for true positives and true negatives, and for pre- and intra-treatment time points. Conclusions and relevance Ct values had limited usefulness for distinguishing between culture-positive and culture-negative cases when field study samples were analyzed. In addition, Ct values were less reliable than fungal culture for determining mycological cure.

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