Development of a Loop-Mediated Isothermal Amplification Assay for Rapid, Sensitive Detection of Campylobacter jejuni in Cattle Farm Samples

Campylobacter jejuni is a leading cause of bacterial foodborne disease worldwide. The detection of this organism in cattle and their environment is important for the control of C. jejuni transmission and the prevention of campylobacteriosis. Here, we describe the development of a rapid and sensitive method for the detection of C. jejuni in naturally contaminated cattle farm samples, based on real-time loop-mediated isothermal amplification (LAMP) of the hipO gene. The LAMP assay was specific (100%inclusivity and exclusivity for 84 C. jejuni and 41 non–C. jejuni strains, respectively), sensitive (detection limit of 100 fg/μl), and quantifiable (R2 = 0.9133). The sensitivity of the LAMP assay was then evaluated for its application to the naturally contaminated cattle farm samples. C. jejuni strains were isolated from 51 (20.7%) of 246 cattle farm samples, and the presence of the hipO gene was tested using the LAMP assay. Amplification of the hipO gene by LAMP within 30 min (mean =10.8 min) in all C. jejuni isolates (n = 51) demonstrated its rapidity and accuracy. Next, template DNA was prepared from a total of 186 enrichment broth cultures of cattle farm samples either by boiling or using a commercial kit, and the sensitivity of detection of C. jejuni was compared between the LAMP and PCR assays. In DNA samples prepared by boiling, the higher sensitivity of the LAMP assay (84.4%) compared with the PCR assay (35.5%) indicates that it is less susceptible to the existence of inhibitors in sample material. In DNA samples prepared using a commercial kit, both the LAMP and PCR assays showed 100% sensitivity. We anticipate that the use of this rapid, sensitive, and simple LAMP assay, which is the first of its kind for the identification and screening of C. jejuni in cattle farm samples, may play an important role in the prevention of C. jejuni contamination in the food chain, thereby reducing the risk of human campylobacteriosis.

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Development and evaluation of a reverse transcription-loop-mediated isothermal amplification assay for rapid and high-sensitive detection of Cryptosporidium in water samples

Water Science & Technology ◽

10.2166/wst.2009.599 ◽

2009 ◽

Vol 60 (8) ◽

pp. 2167-2172 ◽

Cited By ~ 7

Author(s):

A. Inomata ◽

N. Kishida ◽

T. Momoda ◽

M. Akiba ◽

S. Izumiyama ◽

...

Keyword(s):

Reverse Transcription ◽

Water Samples ◽

Cryptosporidium Oocyst ◽

Lamp Assay ◽

Test Tube ◽

Isothermal Amplification ◽

Sensitive Detection ◽

Loop Mediated Isothermal Amplification ◽

Cryptosporidium Oocysts ◽

Amplification Assay

We describe a novel assay for simple, rapid and high-sensitive detection of Cryptosporidium oocysts in water samples using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The detection limit of the developed RT-LAMP assay was as low as 6 × 10−3 oocysts/test tube, which theoretically enables us to detect a Cryptosporidium oocyst and perform duplicated tests even if water samples contain only one oocyst. The developed RT-LAMP assay could more sensitively detect Cryptosporidium oocysts in real water samples than the conventional assay based on microscopic observation.

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Rapid and sensitive detection of Salmonella species targeting the hilA gene using a loop-mediated isothermal amplification assay

Genomics & Informatics ◽

10.5808/gi.21048 ◽

2021 ◽

Vol 19 (3) ◽

pp. e30

Author(s):

Jiyon Chu ◽

Juyoun Shin ◽

Shinseok Kang ◽

Sun Shin ◽

Yeun-Jun Chung

Keyword(s):

Point Of Care ◽

Lamp Assay ◽

Conventional Polymerase Chain Reaction ◽

Isothermal Amplification ◽

Sensitive Detection ◽

Conventional Pcr ◽

Pcr Assay ◽

Loop Mediated Isothermal Amplification ◽

Amplification Assay ◽

Specific Amplification

Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.

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Rapid and Sensitive Detection of Citrus tristeza virus Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay

Methods in Molecular Biology - Citrus Tristeza Virus ◽

10.1007/978-1-4939-9558-5_10 ◽

2019 ◽

pp. 143-150 ◽

Cited By ~ 1

Author(s):

Dilip Kumar Ghosh ◽

Ashish Warghane ◽

Kajal Kumar Biswas

Keyword(s):

Reverse Transcription ◽

Citrus Tristeza Virus ◽

Lamp Assay ◽

Isothermal Amplification ◽

Sensitive Detection ◽

Loop Mediated Isothermal Amplification ◽

Tristeza Virus ◽

Citrus Tristeza

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Development of a Genomics-Based LAMP (Loop-Mediated Isothermal Amplification) Assay for Detection of Pseudomonas fuscovaginae from Rice

Plant Disease ◽

10.1094/pdis-09-13-0957-re ◽

2014 ◽

Vol 98 (7) ◽

pp. 909-915 ◽

Cited By ~ 21

Author(s):

Gavin J. Ash ◽

Jillian M. Lang ◽

Lindsay R. Triplett ◽

Benjamin J. Stodart ◽

Valérie Verdier ◽

...

Keyword(s):

Draft Genome ◽

Lamp Assay ◽

Brown Rot ◽

Isothermal Amplification ◽

Field Surveys ◽

Next Generation Sequencing Technology ◽

Loop Mediated Isothermal Amplification ◽

Pure Cultures ◽

Amplification Assay ◽

Bacterial Plant Pathogen

The vast amount of data available through next-generation sequencing technology is facilitating the design of diagnostic marker systems. This study reports the use of draft genome sequences from the bacterial plant pathogen Pseudomonas fuscovaginae, the cause of sheath brown rot of rice, to describe the genetic diversity within a worldwide collection of strains representing the species. Based on a comparative analysis with the draft sequences, primers for a loop-mediated isothermal amplification (LAMP) assay were developed to identify P. fuscovaginae. The assay reported here reliably differentiated strains of P. fuscovaginae isolated from rice from a range of other bacteria that are commonly isolated from rice and other plants using a primer combination designated Pf8. The LAMP assay identified P. fuscovaginae purified DNA, live or heat-killed cells from pure cultures, and detected the bacterium in extracts or exudates from infected host plant material. The P. fuscovaginae LAMP assay is a suitable diagnostic tool for the glasshouse and laboratory and could be further developed for in-field surveys.

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