Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay

2016 ◽  
Vol 99 (4) ◽  
pp. 1038-1042
Author(s):  
Katie L Korchinski ◽  
Adrian D Land ◽  
David L Craft ◽  
Jennifer L Brzezinski
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Positive Control ◽  
Specific Gene ◽  
Gene Target ◽  
Tobacco Products ◽  
Chewing Tobacco ◽  
Multiplex Reaction ◽  
Internal Positive Control ◽  
Pcr Method

Abstract Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products—including chewing tobacco, pipe tobacco, and snuff—or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

Identification of New PCR Targets and its Validation for Development of Nucleic Acid-based Detection Assay for Melioidosis

2016 ◽  
Vol 1 (1) ◽  
pp. 18
Author(s):  
Sonia Arora ◽  
Duraipandian Thavaselvam ◽  
Archna Prakash ◽  
Ashu Kumar ◽  
Anita Barua ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Cost Effective ◽  
Sybr Green ◽  
Specific Gene ◽  
Pcr Assay ◽  
Gene Target ◽  
Geographical Regions ◽  
Species Specific

Burkholderia pseudomallei the gram negative, soil saprophyte is the causative agent of melioidosis in human and animals. Development of rapid, sensitive, species specific and cost effective molecular assays are needed for detection of B. pseudomallei from clinical and environmental samples and to differentiate it from other closely related bacterial species. In this study, insilico approach was used to identify new species specific gene targets for molecular diagnosis of B. pseudomallei. The identified targets were then analyzed by SYBR Green real time PCR assay for their specificity, sensitivity and presence across different Indian clinical and soil isolates of B. pseudomallei. Out of the three targets studied SYBR Green real time PCR assay targeting bpss0091 gene of B. pseudomallei was found 100% specific, having detection limit of 12.3fg/µl DNA. The bpss0091 gene target was present in all clinical and soil isolates of B. pseudomallei tested thus suggesting bpss0091 gene based SYBR Green real time PCR assay will be useful for detection of B. pseudomallei in different geographical regions.

scholarly journals Detection of Salmonella typhimurium ATCC 14028 in Powder Prepared Traditional Medicines Using Real-Time PCR

2021 ◽  
Vol 4 (3) ◽  
pp. 178-183
Author(s):  
Alfi Sophian ◽  
Ratna Purwaningsih ◽  
Muindar Muindar ◽  
Eka Putri Juniarti Igirisa ◽  
Muhammad Luthfi Amirullah
Keyword(s):  
Salmonella Typhimurium ◽  
Real Time ◽  
Real Time Pcr ◽  
Positive Control ◽  
Negative Control ◽  
Pcr Analysis ◽  
Direct Pcr ◽  
Traditional Medicines ◽  
Pcr Method ◽  
Alternative Testing

The detection of Salmonella typhimurium ATCC 14028 using real-time PCR on powdered traditional medicinal products was carried out in the microbiology and molecular biology testing laboratory of the Food and Drug Administration in Gorontalo. This research aims to provide a reference for alternative testing methods in testing the products of traditional powder preparations on the market. The sample consisted of 10 traditional powder preparations spiked with positive control of S. typhimurium ATCC 14028 phase 2. The method used in the study was real-time PCR analysis using the SYBR® Green method, while DNA isolation using the direct PCR method. Data analysis was performed by analyzing the sample's melting temperature (Tm) curve and comparing it with positive control. The results showed that S. typhimurium ATCC 14028 was detected in samples at an average Tm value of 84.18°C, with ranges of 84.0-84.5°C. For positive control, the Tm value was at 85.2°C, while for the negative control, the Tm value was not detected. Based on these data, it can be concluded that S. typhimurium ATCC 14028 in traditional medicine products powder preparations can be detected using real-time PCR.

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

2018 ◽  
Vol 30 (5) ◽  
pp. 789-792 ◽  
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.

scholarly journals Development and verification of PCR based assay to dectect and quantify garden pea lec gene

2012 ◽  
Vol 30 (No. 3) ◽  
pp. 247-257
Author(s):  
A. Vráblik ◽  
J. Hodek ◽  
K. Demnerová ◽  
J. Soukup ◽  
J. Ovesná
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genetically Modified Organisms ◽  
Initial Point ◽  
Positive Control ◽  
Specific Gene ◽  
Garden Pea ◽  
Pea Lectin ◽  
Lectin Gene ◽  
Species Specific

Genetically modified organisms (GMOs) entering the food chain have became its part, which is necessary to monitor. GMO analyses are used as a control mechanism according to valid acquis communautaire for traceability and labeling of GMOs. Generally, approved PCR based protocols are used and they require stepwise procedures that use amplification of species specific gene as initial point. This study aims to develop and verify PCR based assay for amplification of garden pea lectin gene (Pisum sativum L.) as reference one. Lectin gene was analysed in silico, selected region was amplified and sequenced and new set of species specific primers for identification of garden pea was designed. Conditions of conventional PCR as well as real-time PCR were optimised and specificity of new primer set on DNA extracted from garden pea cultivars as well as DNA extracted from other selected species from Fabaceae family was tested. Quantification of garden pea lectin gene using real-time PCR based on SYBR Green I was optimised and performance characteristics recorded. The characteristics fit to method acceptance criteria range. Plasmid with garden pea lectin sequence was developed and plasmid is available as a positive control.   

scholarly journals A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

2006 ◽  
Vol 6 (1) ◽  
Author(s):  
Livia Di Trani ◽  
Barbara Bedini ◽  
Isabella Donatelli ◽  
Laura Campitelli ◽  
Barbara Chiappini ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Real Time ◽  
Real Time Pcr ◽  
Positive Control ◽  
Influenza Viruses ◽  
Avian Influenza Viruses ◽  
One Step ◽  
Internal Positive Control ◽  
Mgb Probe

scholarly journals Detection of Salmonella typhimurium ATCC 14028 in supplement health product liquid preparation using Real-Time PCR (qPCR)

2020 ◽  
Vol 18 (2) ◽  
Author(s):  
Alfi Sophian ◽  
RATNA PURWANINGSIH ◽  
BERTHA LOLO LUKITA ◽  
ENI CAHYA NINGSIH
Keyword(s):  
Melting Point ◽  
Salmonella Typhimurium ◽  
Real Time ◽  
Real Time Pcr ◽  
Positive Control ◽  
Health Product ◽  
Direct Pcr ◽  
Liquid Preparation ◽  
Pcr Method ◽  
Negative Controls

Abstract. Sophian A, Purwaningsih R, Lukita BL, Ningsih EC. 2018. Detection of Salmonella typhimurium ATCC 14028 in supplement health product liquid preparation using Real-Time PCR (qPCR). Biofarmasi J Nat Prod Biochem 18: 61-65. Detection of Salmonella typhimurium ATCC 14028 using Real-Time PCR (qPCR) on health supplement products was carried out in the microbiology and molecular biology testing laboratory of the Food and Drug Supervisory Office in Gorontalo. The purpose of this study was to provide an alternative testing method reference in the testing of liquid supplement health supplement products in the market. The sample consisted of 35 samples of liquid supplement health supplements spike with positive control of Salmonella typhimurium ATCC 14028 phase 2. The method used in the study was qPCR analysis using the SYBR Green method, whereas DNA isolation using the direct PCR method. Data analysis was performed based on 2 main criteria: (i) Ct (Cycle threshold) analysis, which is looking at the value of the sample Ct and comparing it with controls and (ii) analysis of melting temperature (Tm), which is the melting point at the temperature at which melt occurs and comparing the melting point to the positive control. The results showed that in the sample, Salmonella typhimurium ATCC 14028 was detected at an average Ct value of 14.43, and an average Tm value of 86.05, for the specificity, LOD and positive control tests were all amplified. For negative controls, Ct and Tm values ​​were not detected. Based on these data it can be concluded that real-time PCR (qPCR) can be used to detect Salmonella typhimurium ATCC 14028 in liquid supplement health supplement products.

scholarly journals Design of FRET-TaqMan probes for multiplex real-time PCR using an internal positive control

BioTechniques ◽  
2009 ◽  
Vol 46 (7) ◽  
pp. 519-524 ◽  
Author(s):  
Prithiviraj Jothikumar ◽  
Vincent Hill ◽  
Jothikumar Narayanan
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Positive Control ◽  
Taqman Probes ◽  
Internal Positive Control

Development of an astrovirus RT–PCR detection assay for use with conventional, real-time, and integrated cell culture/RT–PCR

2004 ◽  
Vol 50 (4) ◽  
pp. 269-278 ◽  
Author(s):  
Ann C Grimm ◽  
Jennifer L Cashdollar ◽  
Frederick P Williams ◽  
G Shay Fout
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Water ◽  
Positive Control ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Detection Assay ◽  
Stool Samples ◽  
Pcr Method

Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed and optimized a reverse transcription – polymerase chain reaction (RT–PCR) method that was able to amplify all eight astrovirus serotypes in a single reaction. In addition, a positive control construct was designed so that any inhibitors of this astrovirus assay could be detected. The assay was adapted for use in a real-time PCR assay and the sensitivity of these two methods was compared. The real-time assay was then combined with CaCo2 cell culture to produce an integrated cell culture/RT–PCR (ICC/RT–PCR) assay that was able to detect low levels of astrovirus after an incubation of 7 days or less. Also, the sensitivity of the ICC/RT–PCR assay was compared with RT–PCR alone. The methods were used to detect astrovirus in acute phase illness stool samples as well as in a water sample spiked with astrovirus.Key words: astrovirus, RT–PCR, real-time PCR, ICC/RT–PCR, environmental water.

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