Rapid detection of foot-and-mouth disease virus using a field-portable nucleic acid extraction and real-time PCR amplification platform

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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Molecular epidemiology and phylogenetic analysis of foot and mouth disease virus type ‘O’ and evaluation of its carrier state in cattle of Assam by Real time PCR

Indian Journal of Animal Research ◽

10.18805/ijar.b-3670 ◽

2018 ◽

Author(s):

Sangeeta Baro ◽

Krishna Sharma ◽

Biswajyoti Sharma ◽

Shantanu Tamuly ◽

P. Deka ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Real Time ◽

Disease Virus ◽

Real Time Pcr ◽

Sandwich Elisa ◽

Foot And Mouth Disease ◽

Carrier State ◽

Mouth Disease ◽

Mouth Disease Virus ◽

Foot And Mouth

The molecular epidemiological study of foot-and-mouth disease virus (FMDV) has been carried out from different outbreaks in Assam the present study is based on the nucleotide sequencingof circulating FMDV serotype. The samples were subjected to sandwich ELISA, multiplex-PCR and molecular phylogeny to identify the type species. The phylogenetic analysis of virus sequence revealed similarity with theBangladesh isolates in the major branching pattern. The serotype ‘O’has found to be dominant and responsible for most of the recentoutbreaks.Thepersistence of serotype ‘O’ and cytokines expression of IL-1á, IL-1â, IFN-á, TNF-á in blood of recovered animals were done by Real time PCR. The findings indicated that IL-1á, IFN-á and TNF-á genes were up-regulated upto 3 months post infection but IL-1â found to be down regulated with progression of recovery. The present study thus supports that real-time PCR is a powerful technique for reliable detection of persistent FMDV in recovered animals.

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

Diagnostic Microbiology and Infectious Disease ◽

10.1016/j.diagmicrobio.2015.04.006 ◽

2015 ◽

Vol 82 (4) ◽

pp. 297-302 ◽

Cited By ~ 5

Author(s):

A.E. Greer ◽

M.S. Forman ◽

A. Valsamakis

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assays

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Rapid Detection of Salmonella in Pet Food: Design and Evaluation of Integrated Methods Based on Real-Time PCR Detection

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-210 ◽

2012 ◽

Vol 75 (2) ◽

pp. 347-352 ◽

Cited By ~ 9

Author(s):

PRIYA BALACHANDRAN ◽

MARIA FRIBERG ◽

V. VANLANDINGHAM ◽

K. KOZAK ◽

AMANDA MANOLIS ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Foodborne Pathogens ◽

Real Time Pcr ◽

Rapid Detection ◽

Companion Animals ◽

Magnetic Bead ◽

Acid Extraction ◽

Food Matrix ◽

Pet Food

Reducing the risk of Salmonella contamination in pet food is critical for both companion animals and humans, and its importance is reflected by the substantial increase in the demand for pathogen testing. Accurate and rapid detection of foodborne pathogens improves food safety, protects the public health, and benefits food producers by assuring product quality while facilitating product release in a timely manner. Traditional culture-based methods for Salmonella screening are laborious and can take 5 to 7 days to obtain definitive results. In this study, we developed two methods for the detection of low levels of Salmonella in pet food using real-time PCR: (i) detection of Salmonella in 25 g of dried pet food in less than 14 h with an automated magnetic bead–based nucleic acid extraction method and (ii) detection of Salmonella in 375 g of composite dry pet food matrix in less than 24 h with a manual centrifugation-based nucleic acid preparation method. Both methods included a preclarification step using a novel protocol that removes food matrix–associated debris and PCR inhibitors and improves the sensitivity of detection. Validation studies revealed no significant differences between the two real-time PCR methods and the standard U.S. Food and Drug Administration Bacteriological Analytical Manual (chapter 5) culture confirmation method.

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