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 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 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

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 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

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.

scholarly journals Short Communication: Detection of Salmonella typhimurium ATCC 14028 and Listeria monocytogenes ATCC 7644 in processed meat products using Real-Time PCR Multiplex Method

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Alfi Sophian ◽  
RATNA PURWANINGSIH ◽  
EKA PUTRI JUNIARTI IGIRISA ◽  
MUHAMMAD LUTHFI AMIRULLAH ◽  
BERTHA LOLO LUKITA ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Salmonella Typhimurium ◽  
Real Time ◽  
Melting Temperature ◽  
Real Time Pcr ◽  
Meat Products ◽  
Processed Meat ◽  
Ct Value ◽  
Pcr Multiplex ◽  
Pcr Method

Abstract. Sophian A, Purwaningsih R, Igirisa RPJ, Amirullah ML, Lukita BL, Fitri RA. 2020. Short Communication: Detection of Salmonella typhimurium ATCC 14028 and Listeria monocytogenes ATCC 7644 in processed meat products using Real-Time PCR Multiplex Method. Asian J Nad Prod Biochem 21: 17-20. The detection of Salmonella typhimurium ATCC 14028 and Listeria monocytogenes ATCC 7644 in processed meat products was carried out using Multiplex Real-Time PCR (qPCR) in the Microbiology and Molecular Biology Laboratory at the Indonesian Food and Drug Authority in Gorontalo. The purpose of this study was to provide alternative testing methods for food products circulating in the market. The sample consisted of 25 samples of processed meat products spike with Salmonella typhimurium ATCC 14028 phase 2 and Listeria monocytogenes ATCC 7644 phase 2. The method used in the study was qPCR analysis using the SYBR Green method, while DNA isolation used the direct PCR method. Data analysis was carried out based on Cycle threshold and Melting temperature based on two main criteria. Cycle threshold (Ct) analysis determines the Ct value of the sample and comparing it with the control. Melting temperature (Tm) analysis determines the temperature at which 50% of double-stranded DNA changed to a single standard and comparing it with the melting temperature of positive control. The results showed Salmonella typhimurium ATCC 14028 in the processed meat was detected at an average Ct value of 10.34, and a Tm value of 85.70. The presence of Listeria monocytogenes ATCC 7644 in the samples was recognized at an average Ct value of 14.04, and an average Tm value of 80.07. It can be concluded that the real-time multiplex PCR method can be used to detect Salmonella typhimurium ATCC 14028 and Listeria monocytogenes ATCC 7644 by using the melting curve (Tm) analysis.

Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

2020 ◽  
Vol 18 ◽  
Author(s):  
Pegah Shakib ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
Streptococcus Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Cross Sectional Study ◽  
Laboratory Culture ◽  
Cross Sectional ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

Development and application of a novel reverse transcription real-time PCR method for miR-499 quantification

2013 ◽  
Vol 46 (15) ◽  
pp. 1566-1571 ◽  
Author(s):  
Weidong Zheng ◽  
Yuwei Di ◽  
Yinghong Liu ◽  
Ge Huang ◽  
Youwei Zheng ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Pcr Method
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