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

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.   

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

Defence Life Science Journal ◽

10.14429/dlsj.1.10056 ◽

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.

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

Journal of AOAC International ◽

10.5740/jaoacint.16-0008 ◽

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.

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A real-time PCR method for detection of CpTI (Cowpea Trypsin Inhibitor) gene in the genetically modified rice originating from China

Science and Technology Development Journal ◽

10.32508/stdj.v18i2.1145 ◽

2015 ◽

Vol 18 (2) ◽

pp. 74-86

Author(s):

Linh Thi My Nguyen ◽

Thanh Nguyen Chu ◽

Le Van Bui

Keyword(s):

Real Time ◽

Transgenic Rice ◽

Trypsin Inhibitor ◽

Real Time Pcr ◽

Genetically Modified Organisms ◽

Limit Of Detection ◽

Genetically Modified ◽

Positive Control ◽

Amplification Efficiency ◽

Cowpea Trypsin Inhibitor

Labelling and traceability of genetically modified organisms (GMOs) are necessary for trade and regulation in the world and Vietnam. Cowpea Trypsin Inhibitor (CpTI) gene encodes a trypsin inhibitor which is considered as a suitable candidate for developing insect-resistant transgenic plants, especially transgenic rice lines originating from China. In this study, we established a real-time PCR protocol to detect the CpTI gene in transgenic rice. The protocol with CpTI-F and CpTI-R primers, 300 nM primers, 0.5 X SYBR Green I, annealing temperature at 62 0C showed the best results. Amplification efficiency is 94.64 % and the limit of detection is 50 copies. Moreover, PCR product of CpTI gene was cloned into pBluescript plasmid using as a positive control

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Development and Validation of a P-35S, T-nos, T-35S and P-FMV Tetraplex Real-time PCR Screening Method to Detect Regulatory Genes of Genetically Modified Organisms in Food

CHIMIA International Journal for Chemistry ◽

10.2533/chimia.2014.701 ◽

2014 ◽

Vol 68 (10) ◽

pp. 701-704 ◽

Cited By ~ 4

Author(s):

Albert Eugster ◽

Petra Murmann ◽

Andre Kaenzig ◽

Alda Breitenmoser

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Genetically Modified Organisms ◽

Genetically Modified ◽

Screening Method ◽

Regulatory Genes ◽

Pcr Screening ◽

Development And Validation

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Species-specific real-time PCR cell number quantification of the bloom-forming cyanobacterium Planktothrix agardhii

Archives of Microbiology ◽

10.1007/s00203-012-0809-y ◽

2012 ◽

Vol 194 (9) ◽

pp. 749-757 ◽

Cited By ~ 11

Author(s):

Catarina Churro ◽

Paulo Pereira ◽

Vitor Vasconcelos ◽

Elisabete Valério

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Cell Number ◽

Planktothrix Agardhii ◽

Species Specific

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Detecting Heterobasidion irregulare in Minnesota and Assessment of Indigenous Fungi on Pines

Forests ◽

10.3390/f12010057 ◽

2021 ◽

Vol 12 (1) ◽

pp. 57

Author(s):

Eric Otto ◽

Benjamin Held ◽

Samuel Redford ◽

Robert A. Blanchette

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Wood Decay ◽

Positive Control ◽

Red Pine ◽

Pcr Assay ◽

Nested Polymerase Chain Reaction ◽

Decay Fungi ◽

Field Surveys ◽

Heterobasidion Irregulare

Heterobasidion irregulare is one of the most problematic forest pathogens in the northern hemisphere, but has only been found relatively recently in the north central United States. Discovered in Wisconsin in 1993, but probably established sometime before that, it quickly spread throughout the state. In November 2014, it was found in southeastern Minnesota. Field surveys were then conducted throughout Minnesota with the focus in the southeast near the initial discovery. To find additional infection sites, surveys were conducted with accompanying aerial imagery of red pine (Pinus resinosa Aiton) stands that were previously thinned. Samples were collected from selected sites with dead and dying trees as well as samples from stumps in recently thinned pine stands. These samples were processed first with a nested polymerase chain reaction (PCR) protocol, which was replaced by a real-time PCR assay after its development. No samples tested positive for H. irregulare using these methods and no cultures from isolations were obtained outside the original infection area. Other indigenous fungi were also identified. The majority were wood decay fungi in the Basidiomycota. A spore collection study was also conducted after field surveys. Automated rotary arm spore collectors were used and assayed with an ITS TaqMan real-time PCR assay. Collectors were placed strategically in different areas of Minnesota. A positive control was used in an infected red pine plantation in Wisconsin and this location had the highest number of spores trapped, with 63,776 over a week period. Spores of H. irregulare were detected at several sites in Minnesota, with the highest spore total observed in traps at 413 over a week period. All other locations sampled also had some spores collected except Itasca State Park located in northwestern Minnesota. The weekly deposition of spores ranged from 0 to 1.26 m−2 h−1. Low spore levels occurring in Minnesota indicate that some spores are present, but they are currently being detected in amounts that may not be sufficient for colonization to be successful.

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Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-013-7125-5 ◽

2013 ◽

Vol 405 (21) ◽

pp. 6831-6844 ◽

Cited By ~ 36

Author(s):

Geoffrey Cottenet ◽

Carine Blancpain ◽

Véronique Sonnard ◽

Poh Fong Chuah

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Genetically Modified Organisms ◽

Genetically Modified ◽

Pcr Method ◽

Development And Validation

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Identification of L. casei and L. rhamnosus in the dairy products using Real-Time PCR assay

South Asian Journal of Experimental Biology ◽

10.38150/sajeb.4(5).p222-225 ◽

2015 ◽

Vol 4 (5) ◽

pp. 222-225

Author(s):

K. G. Li ◽

G. P. Pogossian ◽

A. K. Moldagulova ◽

E. E. Bekenova ◽

A. Abdikadirova ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Dairy Products ◽

Production Control ◽

High Efficiency ◽

High Specificity ◽

Industrial Test ◽

Fermented Dairy Products ◽

Species Specific ◽

Fermented Dairy

Lactobacilli are essential and important biological objects used in food pro-duction and medicine. One of the sufficient problems is fast, reliable and highly specific identification of lactobacilli in the scientific research and cur-rent production control. We represent two species-specific real-time PCR in the present study to discriminate L. rhamnosus and L. casei basing on the unique peptidoglycan-hydrolase genes p40 and p75 respectively. PCR pri-mers and probes were designed to provide high specificity discrimination via high temperature of PCR annealing stage. High efficiency of the reactions is provided by the size of amplified DNA fragments minimization. Reliable re-producibility of the target sequences amplification and fluorescence detec-tion provide a basis for the future creation of industrial test-systems for op-erational control in the production of fermented dairy products.

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Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

Journal of Food Protection ◽

10.4315/0362-028x-68.6.1217 ◽

2005 ◽

Vol 68 (6) ◽

pp. 1217-1221 ◽

Cited By ~ 27

Author(s):

PAVEL KRCMAR ◽

EVA RENCOVA

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Dna Sequences ◽

Real Time Pcr ◽

Single Species ◽

Absolute Quantification ◽

Quantitative Detection ◽

Vertebrate Species ◽

Target Species ◽

Species Specific

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

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