scholarly journals Development and Evaluation of the Rapid and Sensitive RPA Assays for Specific Detection of Salmonella spp. in Food Samples

2021 ◽  
Vol 11 ◽  
Author(s):  
Liwei Zhao ◽  
Jianchang Wang ◽  
Xiao Xia Sun ◽  
Jinfeng Wang ◽  
Zhimin Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Foodborne Pathogens ◽  
Real Time Pcr ◽  
Protein A ◽  
Food Samples ◽  
Cross Reactivity ◽  
Salmonella Spp ◽  
Lateral Flow Strip ◽  
The Real ◽  
Conserved Sequence

Salmonella spp. is among the main foodborne pathogens which cause serious foodborne diseases. An isothermal real-time recombinase polymerase amplification (RPA) and lateral flow strip detection (LFS RPA) were used to detect Salmonella spp. targeting the conserved sequence of invasion protein A (invA). The Real-time RPA was performed in a portable florescence scanner at 39°C for 20 min. The LFS RPA was performed in an incubator block at 39°C for 15 min, under the same condition that the amplifications could be inspected by the naked eyes on the LFS within 5 min. The detection limit of Salmonella spp. DNA using real-time RPA was 1.1 × 101 fg, which was the same with real-time PCR but 10 times higher than that of LFS RPA assay. Moreover, the practicality of discovering Salmonella spp. was validated with artificially contaminated lamb, chicken, and broccoli samples. The analyzing time dropped from 60 min to proximately 5–12 min on the basis of the real-time and LFS RPA assays compared with the real-time PCR assay. Real-time and LFS RPA assays’ results were equally reliable. There was no cross-reactivity with other pathogens in both assays. In addition, the assays had good stability. All of these helped to show that the developed RPA assays were simple, rapid, sensitive, credible, and could be a potential point-of-need (PON) test required mere resources.

MicroSEQ® Salmonella spp. Detection Kit Using the Pathatrix® 10-Pooling Salmonella spp. Kit Linked Protocol Method Modification

2014 ◽  
Vol 97 (2) ◽  
pp. 484-491 ◽  
Author(s):  
Jason Wall ◽  
Rick Conrad ◽  
Kathy Latham ◽  
Eric Liu
Keyword(s):  
Real Time ◽  
Study Design ◽  
Foodborne Pathogens ◽  
Real Time Pcr ◽  
Reference Method ◽  
Immunomagnetic Separation ◽  
Pcr Analysis ◽  
Salmonella Spp ◽  
Culture Methods ◽  
Design For All

Abstract Real-time PCR methods for detecting foodborne pathogens offer the advantages of simplicity and quick time to results compared to traditional culture methods. The addition of a recirculating pooled immunomagnetic separation method prior to real-time PCR analysis increases processing output while reducing bothcost and labor. This AOAC Research Institute method modification study validates the MicroSEQ®Salmonella spp. Detection Kit [AOAC Performance Tested Method(PTM) 031001] linked with the Pathatrix® 10-Pooling Salmonella spp. Kit (AOAC PTM 090203C) in diced tomatoes, chocolate, and deli ham. The Pathatrix 10-Pooling protocol represents a method modification of the enrichment portion of the MicroSEQ Salmonella spp. protocol. The results of the method modification were compared to standard cultural reference methods for diced tomatoes, chocolate, and deli ham. All threematrixes were analyzed in a paired study design. An additional set of chocolate test portions was analyzed using an alternative enrichment medium in an unpaired study design. For all matrixes tested, there were no statistically significant differences in the number of positive test portions detected by the modified candidate method compared to the appropriate reference method. The MicroSEQ Salmonella spp. protocol linked with the Pathatrix individual or 10-Pooling procedure demonstrated reliabilityas a rapid, simplified, method for the preparation of samples and subsequent detection of Salmonella in diced tomatoes, chocolate, and deliham.

scholarly journals Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae

2020 ◽  
Author(s):  
Jinfeng Wang ◽  
Ruiwen Li ◽  
Xiaoxia Sun ◽  
Libing Liu ◽  
Xuepiao Hao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Metal Bath ◽  
Lateral Flow Strip ◽  
The Real ◽  
Mycoplasmal Pneumonia ◽  
Mycoplasma Ovipneumoniae

Abstract Background: Mycoplasmal pneumonia is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae is one of major etiological agent causing mycoplasmal pneumonia. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique, and RPA-based diagnostic assays have been described for the detection of different types of pathogens. Results: The RPA assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. ovipneumoniae targeting a conserved region of the 16S rRNA gene. Real-time RPA was performed in a portable florescence scanner at 39 °C for 20 min. LFS RPA was performed in a portable metal bath incubator at 39 °C for 15 min, and the amplicons were visualized with the naked eyes within 5 min on the lateral flow strip. Both assays were highly specific for M. ovipneumoniae , as there were no cross-reactions with other microorganisms tested, especially the pathogens involved in respiratory complex and other mycoplasmas frequently identified in ruminant s . The limit of detection of LFS RPA assay was 1.0×10 1 copies per reaction using a recombinant plasmid containing target gene as template, which is 10 times lower than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 111 clinical sheep nasal swab and fresh lung samples, and M. ovipneumoniae DNA was detected in 29 samples in the real-time RPA, 31 samples in the LFS RPA and 32 samples in the real-time PCR assay. Compared to real-time PCR, the real-time RPA and LFS RPA showed diagnostic specificity of 100% and 98.73%, diagnostic sensitivity of 90.63% and 93.75%, and a kappa coefficient of 0.932 and 0.934, respectively. Conclusions: The developed real-time RPA and LFS RPA assays provide the attractive and promising tools for rapid, convenient and reliable detection of M. ovipneumoniae , especially in resource-limited settings.

scholarly journals SYBR Green Real-Time PCR Used to Detect Celery in Food

2010 ◽  
Vol 93 (5) ◽  
pp. 1530-1536
Author(s):  
Yajun Wu ◽  
Ying Chen ◽  
Bin Wang ◽  
Yunhua Gao ◽  
Liqun Bai ◽  
...  
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection Method ◽  
Food Samples ◽  
Cross Reactivity ◽  
Sybr Green ◽  
Food Sample ◽  
The European Union ◽  
Absolute Detection Limit

Abstract Celery was found to provoke human allergenic response in some countries. Labeling of celery ingredients was required by the European Union, and the threshold set at 10 mg/kg (0.001%). In our study, a celery mannitol transporter (Mat3) gene-based detection method was established by means of SYBR Green real-time PCR technique. No cross-reactivity was found between celery and the other food materials. Absolute detection limit (LODa), relative detection limit (LODr), and practical detection limit (LODp) of the method were determined through experiments on pure celery DNA, DNA mix, and spiked food samples. The method was able to detect 0.001 raw food sample and 0.01 heated food sample. The utility of the method was confirmed by the investigation of 13 commercial foods.

Assessment of Listeria sp. Interference Using a Molecular Assay To Detect Listeria monocytogenes in Food

2016 ◽  
Vol 79 (1) ◽  
pp. 138-143 ◽  
Author(s):  
SANDRA I. ZITTERMANN ◽  
BRENDA STANGHINI ◽  
RYAN SOO SEE ◽  
ROBERTO G. MELANO ◽  
PETER BOLESZCZUK ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Reference Method ◽  
Food Samples ◽  
Pcr Assay ◽  
The Real ◽  
Current Reference ◽  
Listeria Species

ABSTRACT Detection of Listeria monocytogenes in food is currently based on enrichment methods. When L. monocytogenes is present with other Listeria species in food, the species compete during the enrichment process. Overgrowth competition of the nonpathogenic Listeria species might result in false-negative results obtained with the current reference methods. This potential issue was noted when 50 food samples artificially spiked with L. monocytogenes were tested with a real-time PCR assay and Canada's current reference method, MFHPB-30. Eleven of the samples studied were from foods naturally contaminated with Listeria species other than those used for spiking. The real-time PCR assay detected L. monocytogenes in all 11 of these samples; however, only 6 of these samples were positive by the MFHPB-30 method. To determine whether L. monocytogenes detection can be affected by other species of the same genus due to competition, an L. monocytogenes strain and a Listeria innocua strain with a faster rate of growth in the enrichment broth were artificially coinoculated at different ratios into ground pork meat samples and cultured according to the MFHPB-30 method. L. monocytogenes was detected only by the MFHPB-30 method when L. monocytogenes/L. innocua ratios were 6.0 or higher. In contrast, using the same enrichments, the real-time PCR assay detected L. monocytogenes at ratios as low as 0.6. Taken together, these findings support the hypothesis that L. monocytogenes can be outcompeted by L. innocua during the MFHPB-30 enrichment phase. However, more reliable detection of L. monocytogenes in this situation can be achieved by a PCR-based method mainly because of its sensitivity.

Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

2010 ◽  
Vol 4 (2) ◽  
pp. 131-138 ◽  
Author(s):  
Olaya Ruiz-Rueda ◽  
Marçal Soler ◽  
Laia Calvó ◽  
Jesús L. García-Gil
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Food Samples ◽  
Salmonella Spp

scholarly journals Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae

2020 ◽  
Author(s):  
Jinfeng Wang ◽  
Ruiwen Li ◽  
Xiaoxia Sun ◽  
Libing Liu ◽  
Xuepiao Hao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Metal Bath ◽  
Lateral Flow Strip ◽  
The Real ◽  
Mycoplasmal Pneumonia ◽  
Mycoplasma Ovipneumoniae

Abstract Background Mycoplasmal pneumonia is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae is one of major etiological agent causing mycoplasmal pneumonia. It is an urgent need to develop a rapid and accurate method to detect M. ovipneumoniae . Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique, and RPA-based diagnostic assays have been described for the detection of different types of pathogens. Results The RPA assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. ovipneumoniae targeting a conserved region of the 16SrRNA gene. Real-time RPA was performed in a portable florescence scanner at 39 °C for 20 min. LFS RPA was performed in a portable metal bath incubator at 39 °C for 15 min, and the amplicons were visualized with the naked eyes within 5 min on the lateral flow strip. Both assays were highly specific for M. ovipneumoniae , as there were no cross-reactions with other pathogens tested, especially the M. capricolum subsp. capripneumoniae . The limit of detection of LFS RPA assay was 1.0×10 1 copies per reaction using a recombinant plasmid containing target gene as template, which is 10 times higher than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 46 clinical sheep nasal swab and fresh lung samples, and M. ovipneumoniae DNA was detected in 17 samples in the RPA assays and 19 samples in the real-time PCR assay. The real-time RPA and LFS RPA showed diagnostic specificity of 100%, diagnostic sensitivity of 89.47%, and a kappa coefficient of 0.909. Conclusions The developed real-time RPA and LFS RPA assays provide the attractive and promising tools for rapid, convenient and reliable detection of M. ovipneumoniae , especially in resource-limited settings.

scholarly journals DEVELOPMENT OF REAL TIME POLYMERASE CHAIN REACTION FOR DETECTION OF Salmonella typhimurium AND Salmonella enteritidis IN FISH

2013 ◽  
Vol 7 (2) ◽  
pp. 51
Author(s):  
Tuti Hartati Siregar ◽  
Jennifer Elliman ◽  
Leigh Owens
Keyword(s):  
Salmonella Typhimurium ◽  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Coefficient Of Determination ◽  
Biochemical Tests ◽  
Salmonella Spp ◽  
Enrichment Method ◽  
The Real ◽  
Standard Curve

Previously designed endpoint PCR has been adapted for use with real time PCR to detect the presence of Salmonella typhimurium and Salmonella enteritidis  in fish. Optimization of a standard curve in the presence of herring sperm DNA as background matrix indicated that the real time PCR highly efficient with the Pearson coefficient of determination (R2) value = 0.99937 and slope (M) value = -3.44. An enrichment method (overnight culture) significantly increased (p<0.05) the sensitivity of real time PCR. Comparison of real time PCR and the conventional isolation method based on biochemical tests has been conducted. In terms of their sensitivity, real time PCR and the conventional methods are not significantly different in the level of confidence 95%. Both real time PCR with enrichment method and conventional biochemical method can detect the presence of Salmonella spp. in spiked sample. However the direct extraction method was only detecting the presence of Salmonella in higher concentration. While the sensitivity both conventional and real time PCR are similar, the real time PCR has an advantage to detect the pathogen qualitatively and quantitatively depending on processing method.

APPLICATION OF REAL-TIME PRC TECHNIQUE AND REVERSE DOT-BLOT FOR DETECTION THE HIGH RISK TYPES OF HUMAN PAPILLOMAVIRUS CAUSING CERVICAL CANCER

2017 ◽  
pp. 99-103
Author(s):  
Van Bao Thang Phan ◽  
Hoang Bach Nguyen ◽  
Van Thanh Nguyen ◽  
Thi Nhu Hoa Tran ◽  
Viet Quynh Tram Ngo
Keyword(s):  
Cervical Cancer ◽  
High Risk ◽  
Real Time ◽  
Real Time Pcr ◽  
Dot Blot ◽  
The Real ◽  
Dot Blot Assay ◽  
Hpv Types ◽  
High Risk Hpv ◽  
Reverse Dot Blot

Introduction: Infection with HPV is the main cause of cervical cancer. Determining HPV infection and the types of HPV plays an important role in diagnosis, treatment and prognosis of cervicitis/cervical cancer. Aims: Determining proportion of high-risk HPV types and the occurrence of coinfection with multiple HPV types. Methods: 177 women with cervicitis or abnormal Pap smear result were enrolled in the study. Performing the real-time PCR for detecting HPV and the reverse DOT-BLOT assay for determining type of HPV in cases of positive PCR. Results: 7 types of high-risk HPV was dectected, the majority of these types were HPV type 18 (74.6%) and HPV type 16 (37.6%); the proportion of infection with only one type of HPV was 30.4% and coinfection with multiple HPV types was higher (69.6%), the coinfected cases with 2 and 3 types were dominated (32.2% and 20.3%, respectively) and the coinfected cases with 4 and 5 types were rare. Conclusion: Use of the real-time PCR and reverse DOT-BLOT assay can determine the high-risk HPV types and the occurrence of coinfection with multiple HPV types. Key words: HPV type, Reverse DOT-BLOT, real-time PCR,PCR, cervical cancer

scholarly journals Real-Time PCR as an Alternative Technique for Detection of Dermatophytes in Cattle Herds

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1662
Author(s):  
Dominik Łagowski ◽  
Sebastian Gnat ◽  
Aneta Nowakiewicz ◽  
Aleksandra Trościańczyk
Keyword(s):  
Light Microscopy ◽  
Real Time ◽  
Real Time Pcr ◽  
Direct Analysis ◽  
Carrier Status ◽  
The Real ◽  
Detection Techniques ◽  
Direct Light ◽  
Microscopy Analysis ◽  
Cattle Herds

Dermatophytes are filamentous fungi with the ability to digest and grow on keratinized substrates. The ongoing improvements in fungal detection techniques give new scope for clinical implementations in laboratories and veterinary clinics, including the monitoring of the disease and carrier status. The technologically advanced methods for dermatophyte detection include molecular methods based on PCR. In this context, the aim of this study was to carry out tests on the occurrence of dermatophytes in cattle herds using qPCR methods and a comparative analysis with conventional methods. Each sample collected from ringworm cases and from asymptomatic cattle was divided into three parts and subjected to the real-time PCR technique, direct light microscopy analysis, and culture-based methods. The use of the real-time PCR technique with pan-dermatophyte primers detected the presence of dermatophytes in the sample with a 10.84% (45% vs. 34.17%) higher efficiency than direct analysis with light microscopy. Moreover, a dermatophyte culture was obtained from all samples with a positive qPCR result. In conclusion, it seems that this method can be used with success to detect dermatophytes and monitor cowsheds in ringworm cases and carriers in cattle.

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