Combination of Recombinase Polymerase Amplification with SYBR Green I for naked-eye, same-day detection of Escherichia coli O157:H7 in ground meat

Food Control ◽  
2022 ◽  
Vol 132 ◽  
pp. 108494
Author(s):  
Sarah Azinheiro ◽  
Foteini Roumani ◽  
Laura Rodríguez-Lorenzo ◽  
Joana Carvalho ◽  
Marta Prado ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sybr Green ◽  
Sybr Green I ◽  
Recombinase Polymerase Amplification ◽  
Escherichia Coli O157 ◽  
Ground Meat ◽  
Naked Eye

scholarly journals Naked eye detection of theMycobacterium tuberculosiscomplex by recombinase polymerase amplification-SYBR green I assays

2018 ◽  
Vol 33 (2) ◽  
pp. e22655 ◽  
Author(s):  
Nuntita Singpanomchai ◽  
Yukihiro Akeda ◽  
Kazunori Tomono ◽  
Aki Tamaru ◽  
Pitak Santanirand ◽  
...  
Keyword(s):  
Sybr Green ◽  
Sybr Green I ◽  
Recombinase Polymerase Amplification ◽  
Eye Detection ◽  
Naked Eye ◽  
Naked Eye Detection

scholarly journals Rapid detection of multidrug-resistant tuberculosis based on allele-specific recombinase polymerase amplification and colorimetric detection

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0253235
Author(s):  
Nuntita Singpanomchai ◽  
Yukihiro Akeda ◽  
Kazunori Tomono ◽  
Aki Tamaru ◽  
Pitak Santanirand ◽  
...  
Keyword(s):  
Multidrug Resistant ◽  
Sybr Green ◽  
Sybr Green I ◽  
Recombinase Polymerase Amplification ◽  
Low Resource Settings ◽  
Low Resource ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Mdr Tb ◽  
Allele Specific

Multidrug-resistant tuberculosis (MDR-TB) poses a serious threat to TB control. Early diagnosis and proper treatment are essential factors to limit the spread of the disease. The existing molecular tests for MDR-TB usually require specific instruments, steady power supply, and routine maintenance, which might be obstacles for low-resource settings. This study aimed to develop allele-specific isothermal recombinase polymerase amplification (allele-specific RPA) to simultaneously detect the most common mutations in the rpoB gene at codons 516, 526, and 531, which are associated with rifampicin resistance, and in the katG gene at codon 315, which is related to isoniazid resistance. Allele-specific primers targeting four major mutations, rpoB516, rpoB526, rpoB531, and katG315, were constructed and used in individual RPA reactions. The RPA amplicons were endpoints detected by the naked eye immediately after applying SYBR Green I. The optimised RPA assay was evaluated with the Mycobacterium tuberculosis wild-type strain H37Rv and 141 clinical M. tuberculosis isolates. The results revealed that allele-specific RPA combined with SYBR Green I detection (AS-RPA/SYBR) detected these four major mutations with 100% sensitivity and specificity relative to DNA sequencing. The limits of detection for these particular mutations with AS-RPA/SYBR were 5 ng. As a result of the outstanding performance of AS-RPA/SYBR, including its easy setup, speed, lack of a specific instrument requirement, and lack of cross-reaction with other bacteria, this technique may be integrated for the molecular diagnosis of MDR-TB, especially in low-resource settings.

scholarly journals Development of a Directly Visualized Recombinase Polymerase Amplification–SYBR Green I Method for the Rapid Detection of African Swine Fever Virus

2020 ◽  
Vol 11 ◽  
Author(s):  
Shuai Zhang ◽  
Aijun Sun ◽  
Bo Wan ◽  
Yongkun Du ◽  
Yanan Wu ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Sybr Green ◽  
Fever Virus ◽  
Diagnostic Methods ◽  
Sybr Green I ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Field Samples ◽  
Standard Plasmid

African swine fever (ASF) is a lethal disease in swine caused by etiologic African swine fever virus (ASFV). The global spread of ASFV has resulted in huge economic losses globally. In the absence of effective vaccines or drugs, pathogen surveillance has been the most important first-line intervention to prevent ASF outbreaks. Among numerous diagnostic methods, recombinase polymerase amplification (RPA)-based detection is capable of producing sensitive and specific results without relying on the use of expensive instruments. However, currently used gene-specific, probe-based RPA for ASFV detection is expensive and time-consuming. To improve the efficiency of ASFV surveillance, a novel directly visualized SYBR Green I-staining RPA (RPAS) method was developed to detect the ASFV genome. SYBR Green I was added to the amplified RPA products for direct visualization by the naked eye. The sensitivity and specificity of this method were confirmed using standard plasmid and inactivated field samples. This method was shown to be highly specific with a detection limit of 103 copies/μl of ASFV in 15 min at 35°C without any cross-reactions with other important porcine viruses selected. In summary, this method enables direct sample visualization with reproducible results for ASFV detection and hence has the potential to be used as a robust tool for ASF prevention and control.

Detection of the Escherichia coli pathogenic geneeaewith three real-time polymerase chain reaction methods

2007 ◽  
Vol 53 (3) ◽  
pp. 398-403 ◽  
Author(s):  
Joanne  Karen McCrea ◽  
Chenyi Liu ◽  
Lai-King Ng ◽  
Gehua Wang
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Detection Limit ◽  
Real Time ◽  
Turnaround Time ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chain Reaction ◽  
Culture Techniques ◽  
Polymerase Chain

Several real-time polymerase chain reaction (PCR) methods are currently available to rapidly detect the presence of a specific DNA sequence. When used for detection of pathogenic organisms, the turnaround time for PCR-based methods is much lower than for traditional culture techniques. This study compared the sensitivity of three real-time PCR methods when detecting the Escherichia coli pathogenic gene eae to determine which method is most effective in identifying very low levels of the organism. The three methods were used to detect the eae gene over a range of DNA concentrations. The differences in sensitivity were statistically significant (p < 0.05), and SYBR Green I PCR was found to have the lowest detection limit of the three; LUX primers had the highest detection limit. Therefore, using a defined DNA concentration for detecting the eae gene, SYBR Green I is the best alternative.

Selection of In Vivo Expressed Genes of Escherichia coli O157:H7 Strain EDL933 in Ground Meat under Elevated Temperature Conditions

2012 ◽  
Vol 75 (10) ◽  
pp. 1743-1750 ◽  
Author(s):  
ANDREA KROJ ◽  
HERBERT SCHMIDT
Keyword(s):  
Escherichia Coli ◽  
Elevated Temperatures ◽  
Genomic Library ◽  
Transport Processes ◽  
Enterohemorrhagic Escherichia Coli ◽  
Escherichia Coli O157 ◽  
Ground Meat ◽  
E Coli ◽  
Selection Of

Enterohemorrhagic Escherichia coli O157:H7 strains are important foodborne pathogens that are often transmitted to humans by the ingestion of raw or undercooked meat of bovine origin. To investigate adaptation of this pathogen during persistence and growth in ground meat, we established an in vivo expression technology model to identify genes that are expressed during growth in this food matrix under elevated temperatures (42°C). To improve on the antibiotic-based selection method, we constructed the promoter trap vector pAK-1, containing a promoterless kanamycin resistance gene. A genomic library of E. coli O157:H7 strain EDL933 was constructed in pAK-1 and used for promoter selection in ground meat. The 20 in vivo expressed genes identified were associated with transport processes, metabolism, macromolecule synthesis, and stress response. For most of the identified genes, only hypothetical functions could be assigned. The results of our study provide the first insights into the complex response of E. coli O157:H7 to a ground meat environment under elevated temperatures and establish a suitable vector for promoter studies or selection of in vivo induced promoters in foods such as ground meat.

A novel method to detect meat adulteration by recombinase polymerase amplification and SYBR green I

2018 ◽  
Vol 266 ◽  
pp. 73-78 ◽  
Author(s):  
Yuhao Cao ◽  
Kaizhi Zheng ◽  
Junfang Jiang ◽  
Jianliang Wu ◽  
Fangxiong Shi ◽  
...  
Keyword(s):  
Sybr Green ◽  
Sybr Green I ◽  
Recombinase Polymerase Amplification ◽  
Novel Method

Effects of Preparation Methods on the Microbiological Safety of Home-Dried Meat Jerky

2004 ◽  
Vol 67 (10) ◽  
pp. 2337-2341 ◽  
Author(s):  
BRIAN A. NUMMER ◽  
JUDY A. HARRISON ◽  
MARK A. HARRISON ◽  
PATRICIA KENDALL ◽  
JOHN N. SOFOS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Listeria Monocytogenes ◽  
Food Product ◽  
Foodborne Illness ◽  
Escherichia Coli O157 ◽  
Ground Meat ◽  
Preparation Methods ◽  
Home Food ◽  
Whole Muscle

Historically, drying meats to produce jerky was considered to be a safe preservation process and the convenience and flavor of jerky has made it a popular food product for home food preservers. Recent outbreaks of foodborne illness related to both home-dried and commercially manufactured jerky have raised concerns about the safety of the product. Some traditional home recipes and drying processes were shown to be inadequate to destroy Escherichia coli O157, Salmonella, Staphylococcus aureus, and Listeria monocytogenes in both whole-muscle and ground-meat jerky. Several research studies have identified processes such as precooking meats before drying, using acidic marinades, cooking meats after drying, or some combination of these treatments that can destroy pathogens of concern to produce microbiologically safe and palatable meat jerky at home.

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