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 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.

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 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.

scholarly journals Detection of Salmonella typhimurium and Escherichia coli in artificially inoculated Milk sample using Real Time PCR method

2019 ◽  
Vol 1402 ◽  
pp. 055084
Author(s):  
M Nurjayadi ◽  
U R Efrianti ◽  
N Azizah ◽  
F Kurniadewi ◽  
V Saamia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Real Time ◽  
Milk Sample ◽  
Real Time Pcr ◽  
Pcr Method

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.

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.

scholarly journals Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples

2011 ◽  
Vol 77 (18) ◽  
pp. 6570-6578 ◽  
Author(s):  
Sonia E. Létant ◽  
Gloria A. Murphy ◽  
Teneile M. Alfaro ◽  
Julie R. Avila ◽  
Staci R. Kane ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Real Time ◽  
Incubation Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Limit Of Detection ◽  
Turnaround Time ◽  
Pcr Analysis ◽  
Content Type ◽  
Pcr Method

ABSTRACTIn the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulentBacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for theB. anthracischromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulentB. anthracisin environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.

Broad Range Evaluation of the Matrix Solubilization (Matrix Lysis) Strategy for Direct Enumeration of Foodborne Pathogens by Nucleic Acids Technologies

2009 ◽  
Vol 72 (6) ◽  
pp. 1225-1233 ◽  
Author(s):  
EVA MAYRL ◽  
BARBARA ROEDER ◽  
PATRICK MESTER ◽  
MARTIN WAGNER ◽  
PETER ROSSMANITH
Keyword(s):  
Salmonella Typhimurium ◽  
Real Time ◽  
Real Time Pcr ◽  
Sodium Dodecyl ◽  
Ice Cream ◽  
Model Organisms ◽  
Pcr Analysis ◽  
Relative Standard Error ◽  
Bacterial Cells ◽  
Relative Standard

A previously published rapid (<5 h) proof-of-concept protocol for the concentration of the foodborne pathogen Listeria monocytogenes from milk, based on the solubilization of the food matrix, was further evaluated. The original protocol was modified to detect gram-negative and other gram-positive bacteria and to broaden the range of food matrices by using Lutensol instead of sodium dodecyl sulfate as the main detergent in the buffer. A new protocol using a protease and sucrose buffer was established for the analysis of meat and fish. Matrix lysis was used for dairy products, ice cream, milk, fish, meat, eggs, and blood. Solubilization of the foodstuffs resulted in bacterial pellets of reasonable size for further quantification. Using L. monocytogenes, Staphylococccus aureus, Bacillus cereus, Escherichia coli, and Salmonella Typhimurium as model organisms, microscopic analysis of the remaining bacterial pellets revealed that the recovered bacteria remained physically intact, albeit their viability was compromised. In model experiments using free DNA, the free target DNA was reduced by 5 log units. The compatibility of matrix lysis with subsequent real-time PCR analysis has been demonstrated with salmon, chicken, egg, ice cream, cheese, and blood samples that were artificially contaminated with L. monocytogenes, S. aureus, and Salmonella Typhimurium. These experiments resulted in an average recovery of 48.7% (relative standard error, 83.4%) of the inoculated bacterial cells with the real-time PCR assay. The average detection limit of the method was 7.3 CFU/ml for all examined foodstuffs and bacterial target organisms.

scholarly journals Isıl İşlem Görmüş Sütlerde Salmonella Typhimurium Canlı Hücrelerinin PMA/Real-Time PCR ile Belirlenmesi

2017 ◽  
Vol 5 (5) ◽  
pp. 518
Author(s):  
Zülal Kesmen ◽  
Hakiye Aslan
Keyword(s):  
Salmonella Typhimurium ◽  
Real Time ◽  
Real Time Pcr ◽  
False Positive ◽  
Live Cells ◽  
Dead Cell ◽  
Milk Samples ◽  
Heat Treated ◽  
Pcr Method ◽  
Positive Results

Applying different technological processes during the production of food has a lethal effect on the bacteria but DNA of these bacterial strains may cause false positive results when detected by real time PCR technique because they preserve their existence for a certain period of time. To overcome this shortcoming of the real time PCR technique, a new method has been developed in recent years, based on the removal of dead cell DNA from the medium by treatment with Propodium Monoazide (PMA) before DNA extraction. In this study, real-time PCR method was combined with PMA application for the detection of live cells of Salmonella Typhimurium in heat treated milk samples. For this purpose, milk samples inoculated with S. Tyhimurium were heat treated at different temperatures (60, 65, 70 and 75°C) and times (15, 60, 300, 900 sec) and number of live bacteria was determined comparatively by direct real-time PCR, PMA/real-time PCR and conventional cultural method. As a result, unlike the direct real time PCR technique, PMA/real-time PCR method prevents to a certain extent of false positive results from dead cells at all tested temperatures and times but higher results were obtained from PMA/real-time PCR method when compared to conventional cultural results. Therefore, further studies should be carried out to optimize the conditions of the PMA application in order to eliminate the high positive results detected by the PMA / real-time PCR method

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