scholarly journals Development of a Real-time PCR Method Targeting an Unauthorized Genetically Modified Microorganism Producing Alpha-Amylase

2021 ◽  
Author(s):  
Marie-Alice Fraiture ◽  
Ugo Marchesi ◽  
Daniela Verginelli ◽  
Nina Papazova ◽  
Nancy H. C. Roosens
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genetically Modified ◽  
Alpha Amylase ◽  
Pcr Analysis ◽  
Second Line ◽  
Fermentation Products ◽  
Detection Strategy ◽  
Specificity And Sensitivity ◽  
Pcr Method

AbstractUsing a recently developed genetically modified microorganisms (GMM) detection strategy, unexpected contaminations of unauthorized GMM in commercialized microbial fermentation products have been reported. A first-line real-time PCR screening analysis was initially performed to determine the presence of key targets frequently found in genetically modified (GM) bacteria. A second-line real-time PCR analysis was subsequently applied to identify specific GMM, including to date a GM Bacillus velezensis producing protease and a GM B. subtilis producing vitamin B2. In this study, an additional real-time PCR method specific to a newly identified GMM producing alpha-amylase was developed to be integrated in such second-line real-time PCR analysis, allowing to strengthen the GMM detection strategy. This method was successfully validated based on the assessment of its specificity and sensitivity performance. In addition, its applicability was confirmed using several food enzyme products commercialized on the market. Finally, via its transfer to an external laboratory, the transferability of the in-house validated method was positively evaluated, allowing its easy implementation in enforcement laboratories.

Interlaboratory Validation Study of an Event-Specific Real-time Polymerase Chain Reaction Detection Method for Genetically Modified 55-1 Papaya

2013 ◽  
Vol 96 (5) ◽  
pp. 1054-1058
Author(s):  
Akio Noguchi ◽  
Kosuke Nakamura ◽  
Kozue Sakata ◽  
Tomoko Kobayashi ◽  
Hiroshi Akiyama ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection Method ◽  
Genetically Modified ◽  
Pcr Analysis ◽  
Labeling Regulations ◽  
Interlaboratory Validation ◽  
Endogenous Reference ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Genetically modified (GM) papaya line 55-1 (55-1) is resistant to papaya ringspot virus infection, and is commercially available in several countries. A specific detection method for 55-1 is required for mandatory labeling regulations. An event-specific real-time PCR method was developed by our laboratory. To validate the method, interlaboratory validation of event-specific qualitative real-time PCR analysis for 55-1 was performed in collaboration with 12 laboratories. DNA extraction and real-time PCR reaction methods were evaluated using 12 blind samples: six non-GM papayas and six GM papayas in each laboratory. Genomic DNA was highly purified from all papayas using an ion-exchange column, and the resulting DNA sample was analyzed using real-time PCR. Papaya endogenous reference gene chymopapain (CHY) and the event-specific 55-1 targeted sequence were detected in GM papayas whereas CHY alone was detected in non-GM papayas in all laboratories. The cycle threshold values of CHY and the 55-1 targeted sequence showed high repeatability (RSDr 0.6–0.8%) and reproducibility (RSDR 2.2–3.6%). This study demonstrates that the 55-1 real-time PCR detection method is a useful and reliable method to monitor 55-1 papaya in foods.

scholarly journals Evaluation of a Real Time PCR Assay Method for the Detection of Genetically Modified Organisms in Food Products

2020 ◽  
Vol 9 (2) ◽  
pp. 1
Author(s):  
Eleni Spanea ◽  
Theofania Tsironi ◽  
Efstathia Tsakali ◽  
Anthimia Batrinou ◽  
Valentina Stefanou ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Food Products ◽  
Assay Method ◽  
Genetically Modified Maize ◽  
Nos Terminator ◽  
Repeatability And Reproducibility ◽  
Pcr Method

The objective of the study was to determine qualitatively by validated Real Time PCR method the occurrence of genetically modified maize and soybean in commercial food products from the Greek market. 70 independent samples were collected, including products from different categories (i.e. cereal based, biscuits and snacks) which declared either corn or soybean on the labelling. The result of the study indicated that 37.1% of maize and soy products (n=70) displayed in the Greek market have detectable levels of genetically modified maize or soy. These products were identified by specific primers and included common GMΟ detection primers for 35S and NOS terminator. Adequate repeatability and reproducibility was demonstrated for the applied Real Time PCR method, as evaluated by intra- and inter-laboratory tests.

Collaborative trial validation of a construct-specific real-time PCR method for detection of genetically modified linseed event ‘CDC Triffid’ FP967

2011 ◽  
Vol 232 (3) ◽  
pp. 557-561 ◽  
Author(s):  
Lutz Grohmann ◽  
Ulrich Busch ◽  
Sven Pecoraro ◽  
Norbert Hess ◽  
Klaus Pietsch ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genetically Modified ◽  
Collaborative Trial ◽  
Pcr Method

Crop Plant Genotyping by Real-Time PCR Analysis of Crude Extracts of Seeds

2015 ◽  
Vol 98 (1) ◽  
pp. 1-4 ◽  
Author(s):  
Yang Lan ◽  
Cheng-En Wang ◽  
Chuan-Fang Wu ◽  
Zhong-Yu Liu ◽  
Chun-Fang Gao
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Single Copy ◽  
Crop Plants ◽  
Crop Plant ◽  
Pcr Analysis ◽  
Melting Curve Analysis ◽  
Pcr Method ◽  
Direct Amplification

Abstract Development of agricultural biotechnology requires rapid and convenient methods for crop plant genotyping. Real-time PCR is sensitive and reliable, and has been a routine technique in plant research. However, its application is limited by the cumbersome DNAtemplate preparation procedures. We tested three PCRmaster mixes for direct amplification of crude seed DNA extracts without extensive purification. One mixhad higher resistance to plant-derived PCR inhibitors and was shown to be applicable to various important crop plants. Furthermore, this method is capable of detecting single-copy genes from 2 mg pieces of seeds repetitively. Meanwhile, melting curve analysis could detect amplicons directly without electrophoresis manipulations. Taken together, this direct real-time PCR method provides a rapid and convenient toolfor seed genotypic screening in crop plants.

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.

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