Comparison of BKV quantification using a single automated nucleic acid extraction platform and 3 real-time PCR assays

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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Limited-Resource Preparable Chitosan Magnetic Particles for Extracting Amplification-Ready Zeptomole-Range Nucleic Acid from Complex Biofluid

10.26434/chemrxiv.14579127 ◽

2021 ◽

Author(s):

Sayantan Tripathy ◽

Arunansu Talukdar ◽

Goutam Pramanik ◽

P. V. Rajesh ◽

Souradyuti Ghosh

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Magnetic Particles ◽

Limited Resource ◽

Nucleic Acid Amplification ◽

Acid Extraction ◽

Analytical Sensitivity ◽

Nucleic Acid Extraction ◽

Spin Column

<b>Layman Summary: </b>Nucleic acid extraction is a key prerequisite for any nucleic acid amplification test (NAAT) or isothermal NAAT (iNAAT) based molecular diagnosis assays.<b> </b>Existing methods utilizes spin column system for nucleic acid extraction which are unsuitable for limited resource settings. Our work explores two methods for chitosan coated magnetic particle preparation that can be executed within 6 h from commonly available chemicals with nothing but a magnetic stirrer and water bath and doable by a minimally trained person. We will also investigated the compatibility of the extracted nucleic acid with downstream NAATs such as real time LAMP, colorimetric LAMP, and real time PCR. In the process, we established the analytical sensitivity of the overall method.<div><br><div><b>Characterization methods</b>: SEM, XRD, EDX, FT-IR</div><div><br></div><div><b>Bioanalytical methods:</b> Real time LAMP, Colorimetric LAMP, Real time PCR</div></div>

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High-throughput HTLV-1 proviral DNA detection system using a nucleic acid extraction robot and real-time PCR detection.

Journal of the Japan Society of Blood Transfusion ◽

10.3925/jjtc1958.47.378 ◽

2001 ◽

Vol 47 (3) ◽

pp. 378-383 ◽

Cited By ~ 1

Author(s):

Chieko Matsumoto ◽

Rieko Shiozawa ◽

Shigeki Mitsunaga ◽

Akiko Ichikawa ◽

Rika Ishiwatari ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

High Throughput ◽

Real Time Pcr ◽

Detection System ◽

Dna Detection ◽

Pcr Detection ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Proviral Dna

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Development and application of a canine endogenous internal positive control for use in real-time PCR assays

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638718795206 ◽

2018 ◽

Vol 30 (5) ◽

pp. 789-792 ◽

Cited By ~ 2

Author(s):

Joseph J. Modarelli ◽

Pamela J. Ferro ◽

Maria D. Esteve-Gasent

Keyword(s):

Real Time ◽

Real Time Pcr ◽

False Negative ◽

Positive Control ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Negative Results ◽

False Negative Results ◽

Internal Positive Control ◽

Pcr Assays

Real-time PCR (rtPCR) tests have become a method of choice in many diagnostic settings, both animal and human. A concern remains, however, regarding rtPCR assay inhibition during nucleic acid extraction and/or rtPCR reaction process that may result in false-negative results. The use of an internal positive control, either endogenous or exogenous, to mitigate this issue has become more commonplace. We identified and standardized an endogenous internal positive control that can be utilized in rtPCR assays targeting canine-specific pathogens in either a singleplex or multiplex format. The target chosen for the endogenous internal positive control (EIPC-K9) was a highly conserved region in canine mitochondrial DNA. Samples from 240 dogs and 11 other species were screened with EIPC-K9; all canine samples were detected, and no cross-amplification with other species tested was observed. Additionally, no inhibition was noted when comparing singleplex to multiplex rtPCR formats.

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