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

2009 ◽  
Vol 60 (8) ◽  
pp. 2167-2172 ◽  
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.

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

2014 ◽  
Vol 77 (9) ◽  
pp. 1593-1598 ◽  
Author(s):  
HEE-JIN DONG ◽  
AE-RI CHO ◽  
TAE-WOOK HAHN ◽  
SEONGBEOM CHO
Keyword(s):  
Campylobacter Jejuni ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Sensitive Detection ◽  
Cattle Farm ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
Pcr Assays ◽  
Template Dna ◽  
Commercial Kit

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.

scholarly journals Rapid and Sensitive Detection of Norovirus Genomes in Oysters by a Two-Step Isothermal Amplification Assay System Combining Nucleic Acid Sequence-Based Amplification and Reverse Transcription-Loop-Mediated Isothermal Amplification Assays

2008 ◽  
Vol 74 (12) ◽  
pp. 3912-3914 ◽  
Author(s):  
Shinji Fukuda ◽  
Yukie Sasaki ◽  
Masato Seno
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Isothermal Amplification ◽  
Assay System ◽  
Sensitive Detection ◽  
Nucleic Acid Sequence ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
Seminested Pcr

ABSTRACT We developed a two-step isothermal amplification assay system, which achieved the detection of norovirus (NoV) genomes in oysters with a sensitivity similar to that of reverse transcription-seminested PCR. The time taken for the amplification of NoV genomes from RNA extracts was shortened to about 3 h.

scholarly journals Development and Preliminary Evaluation of a Loop-Mediated Isothermal Amplification Procedure for Sensitive Detection of Cryptosporidium Oocysts in Fecal and Water Samples

2007 ◽  
Vol 73 (17) ◽  
pp. 5660-5662 ◽  
Author(s):  
Panagiotis Karanis ◽  
Oriel Thekisoe ◽  
Klytaimnistra Kiouptsi ◽  
Jerry Ongerth ◽  
Ikuo Igarashi ◽  
...  
Keyword(s):  
Diagnostic Tool ◽  
Water Samples ◽  
Epidemiologic Studies ◽  
Isothermal Amplification ◽  
Sensitive Detection ◽  
Preliminary Evaluation ◽  
Fecal Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Cryptosporidium Oocysts ◽  
Amplification Procedure

ABSTRACT A loop-mediated isothermal amplification (LAMP) procedure for the detection of Cryptosporidium in environmental and fecal samples was developed and evaluated. This is the first demonstration of LAMP applied to detection of Cryptosporidium. Due to its specificity and simplicity, the method could become a useful diagnostic tool for epidemiologic studies of Cryptosporidium presence.

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

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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