Multiplex PCR assay and lyophilization for detection of Salmonella spp., Staphylococcus aureus and Bacillus cereus in pork products

Arunrut, Narong; Kiatpathomchai, Wansika; Ananchaipattana, Chiraporn

doi:10.1007/s10068-017-0286-9

Cited by 26 publications

(10 citation statements)

References 20 publications

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“…Furthermore, it has also been reported that gene62181533 amplification can be effectively used to specifically detect the contamination of pork products by Salmonella spp. without cross‐reacting with several other common bacteria (Arunrut et al, ). Thus, based on our results and previous findings, it can be established that gene62181533 is a suitable target gene for the direct detection of Salmonella in biological samples.…”

Section: Discussionmentioning

confidence: 99%

“…Generally, detection and identification of Salmonella spp. is performed by conventional culture methods that are both timeconsuming and labor-intensive, with a minimum of 5 days required to complete the analysis (Ananchaipattana, Bari, & Inatsu, 2016;Arunrut, Kiatpathomchai, & Ananchaipattana, 2018). Immunological assays require less time but provide poor specificity with high rates of false positives.…”

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confidence: 99%

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Development and evaluation of real‐time loop mediated isothermal amplification assay for rapid and sensitive detection of Salmonella spp. in chicken meat products

Arunrut

Kiatpathomchai

Ananchaipattana

2018

Journal of Food Safety

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Salmonella spp., one of the most prevalent bacterial causes of foodborne disease, is a major public health concern due to its common occurrence in chicken meat products. The detection of Salmonella spp. using conventional culturing methods requires an excessive amount of time and effort. Thus, in response to the need for a rapid, sensitive, and convenient method for Salmonella detection, a quantitative assay using real‐time Loop‐mediated isothermal amplification (real‐time LAMP) was developed using gene62181533 as the target sequence amplified. The real‐time LAMP assay does not show cross‐reactivity with several other common bacterial pathogens and the detection limit for genomic DNA in pure culture was found to be 1.2 CFU/ml. Testing with spiked samples simultaneously detected 7 CFU/ml of Salmonella pathogen in the artificially inoculated samples after enrichment for 6 hr. In comparison with the standard culture‐based methods in the analysis of 120 raw chicken meat samples, the results of the sensitivity, accuracy, and specificity tests of the real‐time LAMP assay were 94.02, 90.83, and 86.79%, respectively. These results indicate that this method is not only sensitive and specific but can also be used for rapid detection and differentiation of foodborne disease‐causing bacteria. Practical applications Salmonella is an important bacterial genus which causes most of the common foodborne illnesses worldwide. The disease mainly caused by Salmonella spp. through consumption of contaminated eggs and poultry products is salmonellosis which can manifest as bacterial diarrhea through to septicemia. The result indicates the potential usefulness of real‐time LAMP assay based on the gene62181533 for a rapid, specific, sensitive, and quantitative assay for Salmonella detection in food products. This assay offers a low cost quantitative method in the clinical diagnosis of Salmonella in resource‐limited health‐care facilities and clinical laboratories in developing countries and in field tests.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Development and evaluation of real‐time loop mediated isothermal amplification assay for rapid and sensitive detection of Salmonella spp. in chicken meat products

Arunrut

Kiatpathomchai

Ananchaipattana

2018

Journal of Food Safety

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“…Several publications on freeze-dried PCR mixes have been reported during the past 20 years ( Table S1 ) [ [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] ]. In previous studies, electrophoresis has frequently been used to evaluate the activities of freeze-dried PCR reagents [ 9 , 10 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

Transferable, easy-to-use and room-temperature-storable PCR mixes for microfluidic molecular diagnostics

Wang

et al. 2021

Talanta

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As the outbreak of coronavirus disease 2019 (COVID-19), on-site molecular diagnosis is becoming increasingly important. In this study, a freeze-drying method was introduced for PCR reagents to meet the requirements of microfluidic molecular diagnosis. Using this method, PCR components were pre-mixed and freeze-dried as a bead, which could be transferred into microfluidic chips easily. As this bead only required reconstitution in water, operational steps of PCR were simplified, pipetting errors and errors associated with improper handling of wet reagents could also be reduced. In addition, 19 PCR mixes for different targets (including both RNA and DNA) detection were stable when stored at room temperature (18–25 °C) for 1–2 years and may be stored longer as activity monitoring remains ongoing. To shorten the stability testing time, accelerated stability testing at higher temperatures was proposed. The evaluation periods of the freeze-dried PCR mixes were shortened to less than one month when stored at 56 °C and 80 °C. When attempts were further tried to predict the shelf lives for freeze-dried PCR mixes, our findings challenged the classic view of the Q 10 method as a prediction model for freeze-dried PCR mixes and confirmed for the first time that this prediction was influenced by different factors at varying degrees. These studies and findings are important for the development of molecular diagnosis at both central laboratories and resource-limited areas.

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“…However, lyophilisation process is costly and requires the addition of excipients, such as cryoprotectants and bulking agents. 17,19…”

Section: Introductionmentioning

confidence: 99%

A Novel Air-Dried Multiplex High Resolution Melt Assay for the Detection of Extended Spectrum Beta-Lactamase and Carbapenemase Genes

Atienzar

Williams

Karkey

et al. 2021

Preprint

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Here we describe the development and evaluation of a novel an air-dried high-resolution melt (HRM) assay to detect eight major extended spectrum beta-Lactamase (ESBL) (SHV and CTXM groups 1 and 9) and Carbapenemase (NDM, IMP, KPC, VIM and OXA-48) genes that cause antimicrobial resistance. The assay was evaluated using 440 DNA samples extracted from bacterial isolates from Nepal, Malawi and UK and 390 clinical Enterobacteriaceae isolates with known resistance phenotypes from Nepal. The sensitivity and specificity for detecting the ESBL and Carbapenemase genes in comparison to the reference gel-base PCR and sequencing was 94.7% (95%CI: 92.5%-96.5%) and 99.2% (95%CI: 98.8%-99.5%) and 98.5% (95%CI: 97.0%-99.4%) and 98.5% (95%CI: 98.0%-98.9%) when compared to the original wet format. The overall phenotypic agreement was 91.1% (95%CI: 90.0%-92.9%) on predicting resistance to cefotaxime and carbapenems. We observed good inter-machine reproducibility of the air-dried HRM assay using the Rotor-Gene Q, QuantStudioTM 5, CFX96, LightCycler 480 and MIC. Assay stability upon storage in the fridge, room temperature and oven were assessed at six time points for eight months and no loss of sensitivity occurred under all conditions. We present here a ready-to-use air-dried HRM-PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and Carbapenamase genes to improve AMR diagnosis and treatment.

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Multiplex PCR assay and lyophilization for detection of Salmonella spp., Staphylococcus aureus and Bacillus cereus in pork products

Cited by 26 publications

References 20 publications

Development and evaluation of real‐time loop mediated isothermal amplification assay for rapid and sensitive detection of Salmonella spp. in chicken meat products

Development and evaluation of real‐time loop mediated isothermal amplification assay for rapid and sensitive detection of Salmonella spp. in chicken meat products

Transferable, easy-to-use and room-temperature-storable PCR mixes for microfluidic molecular diagnostics

A Novel Air-Dried Multiplex High Resolution Melt Assay for the Detection of Extended Spectrum Beta-Lactamase and Carbapenemase Genes

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