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

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

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-056 ◽

2014 ◽

Vol 77 (9) ◽

pp. 1593-1598 ◽

Cited By ~ 20

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.

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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 Lawsonia Intracellularis In Pigs By Real-Time Loop-Mediated Isothermal Amplification

Acta veterinaria ◽

10.1515/acve-2015-0002 ◽

2015 ◽

Vol 65 (1) ◽

pp. 20-29 ◽

Cited By ~ 3

Author(s):

PARK Byung-Yong ◽

SHIM Kwan-Seob ◽

KIM Won-Il ◽

HOSSAIN Md Mukter ◽

KIM Bumseok ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Lamp Assay ◽

Isothermal Amplification ◽

Sensitive Detection ◽

Detection Methods ◽

Conventional Pcr ◽

Fecal Samples ◽

Loop Mediated Isothermal Amplification ◽

Time Loop

Abstract A simple and rapid real-time loop-mediated isothermal amplification (LAMP) assay designed to detect Lawsonia (L.) intracellularis, an important bacteria causing proliferative enteropathy in pigs. A set of four primers targeting the ubiquinone/menaquinone biosynthesis methylase (ubiE) gene was designed for the LAMP reaction. Additionally, serial 10-fold dilutions of cultured L. intracellularis and spiked feces were also used for the optimization of real-time LAMP. The lower limit of the linear range of the assay in L. intracellularis was 1.0 × 100 L. intracellularis. Real-time LAMP was 10 and 100 times more sensitive than real-time PCR and conventional PCR detection methods, respectively. Based on testing of 213 porcine fecal samples using real-time LAMP, realtime PCR and PCR, the agreement quotients of real-time LAMP with conventional PCR and with real-time PCR were 0.77 and 0.95, respectively. This study demonstrated that real-time LAMP was a powerful tool for the rapid and sensitive detection of L. intracellularis in porcine fecal samples.

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Development of a Loop-Mediated Isothermal Amplification Assay for the Detection of Dickeya spp.

Phytopathology ◽

10.1094/phyto-04-17-0160-r ◽

2017 ◽

Vol 107 (11) ◽

pp. 1339-1345 ◽

Cited By ~ 10

Author(s):

Jarred Yasuhara-Bell ◽

Glorimar Marrero ◽

Mohammad Arif ◽

Asoka de Silva ◽

Anne M. Alvarez

Keyword(s):

Lamp Assay ◽

Conventional Polymerase Chain Reaction ◽

Isothermal Amplification ◽

Northern Europe ◽

Potato Seed ◽

Chain Reaction ◽

Bacterial Populations ◽

Loop Mediated Isothermal Amplification ◽

Amplification Assay ◽

Polymerase Chain

Dickeya and Pectobacterium spp. are responsible for soft-rotting diseases of several plant species, some with overlapping host range. On potato, symptoms caused by these pathogens cannot be clearly differentiated. Disease results in the downgrading and rejection of potato seed, thus requiring additional phytosanitary restrictions across Northern Europe and other parts of the world. In an effort to provide a more timely and accurate diagnostic to distinguish these two groups of pathogens, a method for detecting Dickeya spp. using loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay can be used to test crude extracts prepared directly from symptomatic lesions. The entire test can be completed in less than 30 min, making it faster than the current diagnostic standard, the pelADE conventional polymerase chain reaction. Additionally, the LAMP assay was able to detect Dickeya DNA in samples spiked with varying amounts of Pectobacterium DNA, thus demonstrating the highly specific and sensitive nature of the assay, which can be applied on survey samples with mixed soft-rotting bacterial populations.

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Rapid detection of Pecan Root-Knot Nematode, Meloidogyne partityla, in laboratory and field conditions using loop-mediated isothermal amplification

10.1101/2020.01.09.900076 ◽

2020 ◽

Author(s):

Sumyya Waliullah ◽

Jessica Bell ◽

Tammy Stackhouse ◽

Ganpati Jagdale ◽

Abolfazl Hajihassani ◽

...

Keyword(s):

Lamp Assay ◽

Nematode Species ◽

Conventional Polymerase Chain Reaction ◽

Cross Reactivity ◽

Isothermal Amplification ◽

Field Conditions ◽

Lamp Method ◽

Conventional Pcr ◽

Root Knot Nematode ◽

Loop Mediated Isothermal Amplification

AbstractMeloidogyne partityla is the dominant root-knot nematode (RKN) species parasitizing pecan in Georgia. This species is known to cause a reduction in root growth and a decline in yields from mature pecan trees. Rapid and accurate diagnosis of this RKN is required to control this nematode disease and reduce losses in pecan production. In this study, a loop-mediated isothermal amplification (LAMP) method was developed for simple, rapid and on-site detection of M. partityla in infested plant roots and validated to detect the nematode in laboratory and field conditions. Specific primers were designed based on the sequence distinction of internal transcribed spacer (ITS)-18S/5.8S ribosomal RNA gene between M. partityla and other Meloidogyne spp. The LAMP detection technique could detect the presence of M. partityla genomic DNA at a concentration as low as 1 pg, and no cross reactivity was found with DNA from other major RKN species such as M. javanica, M. incognita and M. arenaria, and M. hapla. We also conducted a traditional morphology-based diagnostic assay and conventional polymerase chain reaction (PCR) assay to determine which of these techniques was less time consuming, more sensitive, and convenient to use in the field. The LAMP assay provided more rapid results, amplifying the target nematode species in less than 60 min at 65°C, with results 100 times more sensitive than conventional PCR (~2-3 hrs). Morphology-based, traditional diagnosis was highly time-consuming (2 days) and more laborious than conventional PCR and LAMP assays. These features greatly simplified the operating procedure and made the assay a powerful tool for rapid, on-site detection of pecan RKN, M. partityla. The LAMP assay will facilitate accurate pecan nematode diagnosis in the field and contribute to the management of the pathogen.

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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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