scholarly journals Loop-Mediated Isothermal Amplification Assay for Detection of Generic and Verocytotoxin-ProducingEscherichia coliamong Indigenous Individuals in Malaysia

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Cindy Shuan Ju Teh ◽  
Kek Heng Chua ◽  
Yvonne Ai Lian Lim ◽  
Soo Ching Lee ◽  
Kwai Lin Thong
Keyword(s):  
Large Scale ◽  
Lamp Assay ◽  
Asymptomatic Infection ◽  
Isothermal Amplification ◽  
Indigenous Populations ◽  
Bacterial Strains ◽  
E Coli ◽  
Loop Mediated Isothermal Amplification ◽  
Threshold Time ◽  
Amplification Assay

We have successfully developed a Loop-mediated isothermal amplification(LAMP) assay that could specifically detect genericEscherichia coli(E. coli). This assay was tested on 85 bacterial strains and successfully identified 54E. colistrains (average threshold time, Tt = 21.26). The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 102 CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 103 CFU/mL (Tt = 31.12). We have also detected 46 genericE. colifrom 50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producingE. coli(VTEC) positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6 : 1. Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years. The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay.

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

Plant Disease ◽  
2014 ◽  
Vol 98 (7) ◽  
pp. 909-915 ◽  
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.

A sequence-specific plasmonic loop-mediated isothermal amplification assay with orthogonal color readouts enabled by CRISPR Cas12a

2020 ◽  
Vol 56 (24) ◽  
pp. 3536-3538 ◽  
Author(s):  
Rongxing Zhou ◽  
Yongya Li ◽  
Tianyu Dong ◽  
Yanan Tang ◽  
Feng Li
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Complementary Color ◽  
Amplification Assay

CRISPR Cas12a enables a sequence-specific plasmonic LAMP assay with dual complementary color readouts.

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 Development of a Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of the tdh and trh Genes of Vibrio parahaemolyticus and Related Vibrio Species

2009 ◽  
Vol 76 (3) ◽  
pp. 820-828 ◽  
Author(s):  
Wataru Yamazaki ◽  
Yuko Kumeda ◽  
Naoaki Misawa ◽  
Yoshitsugu Nakaguchi ◽  
Mitsuaki Nishibuchi
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Virulence Determinants ◽  
Bacterial Strains ◽  
Phenotypic Differences ◽  
Loop Mediated Isothermal Amplification ◽  
Vibrio Mimicus ◽  
Thermostable Direct Hemolysin

ABSTRACT Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.

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.

A Rapid and Sensitive Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Indian Citrus Ringspot Virus

Plant Disease ◽  
2020 ◽  
Author(s):  
Amol Kokane ◽  
Sunil Kokane ◽  
Ashish Warghane ◽  
Mrugendra G Gubyad ◽  
Ashwani Kumar Sharma ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Color Change ◽  
Lamp Assay ◽  
Single Step ◽  
Isothermal Amplification ◽  
Ringspot Virus ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Field Samples

Indian citrus ringspot virus (ICRSV) is a devastating pathogen that has a particularly deleterious effect on the ‘Kinnow mandarin’, a commercial citrus crop cultivated in the north-west of India. ICRSV belongs to the Mandarivirus genus within the family of Alphaflexiviridae and has a positive sense single-stranded RNA (ssRNA) genome consisting of six open reading frames (ORFs). Severe cases of ICRSV result in a significant reduction in both the yield and quality of crops. Consequently, there is an urgent need to develop methods to detect ICRSV in an accurate and timely manner. Current methods involve a two-step reverse transcriptase-polymerase chain reaction (RT-PCR) that is time-consuming. Here, we describe a novel, one-step, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method for the sensitive and rapid detection of ICRSV. The RT-LAMP assay was standardized by designing and testing four different primers that targeted the coat protein gene of ICRSV. Amplification results were visualized by a color change after addition of SYBR Green I. The standardized RT-LAMP assay was highly specific and successfully detected all 35 ICRSV isolates tested from the Punjab and Haryana states of India. Furthermore, there was no cross-reaction with 17 isolates of five other citrus pathogens that are common in India. ICRSV-RT-LAMP assay developed in the present study is a simple, rapid, sensitive, and specific, technique. Moreover, the assay consists of only a single step and is more cost-effective than existing methods. This represents the first application of RT-LAMP for the detection of ICRSV. Our RT-LAMP assay is a powerful tool for the detection of ICRSV and will be particularly useful for large scale indexing of field samples in diagnostic laboratories, nurseries, and for quarantine applications.

scholarly journals Earlyin plantadetection ofXanthomonas axonopodispv.punicaein pomegranate using enhanced loop-mediated isothermal amplification assay

2018 ◽  
Author(s):  
M.K. PrasannaKumar ◽  
P. Buela Parivallal ◽  
C. Manjunath ◽  
H.B. Mahesh ◽  
Karthik S Narayan ◽  
...  
Keyword(s):  
Post Infection ◽  
Bacterial Blight ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Economic Losses ◽  
Specific Primers ◽  
Loop Mediated Isothermal Amplification ◽  
Negative Results ◽  
Hydroxynaphthol Blue ◽  
Amplification Assay

AbstractBacterial blight in pomegranate caused byXanthomonas axonopodispv.punicae(Xap) is an increasing threat for pomegranate cultivation in India. To prevent the economic losses, it is pivotal to detect the infection in latent stages rather than in later stages. We have developed an enhanced method termed as loop-mediated isothermal amplification (LAMP) technique to evaluate for the latent detection of Xap in pomegranate using six set of specific primers. Three DNA intercalating dyes were used, such as Ethidium bromide, hydroxynaphthol blue (HNB) and SYBR Green resulted in visualising the positivity for LAMP assay. The reaction time and temperature were to be 65°C from 30 min onwards, for the dyes and its sensitivity was observed up to 10−7ng in the LAMP assay. For field applicability, LAMP assay detected Xap on 7thday post infection while the PCR amplified Xap after 11thday post infection. Finally, the specificity of LAMP assay was validated to be positive with ten Xap isolates for its accuracy and 29 non-Xap bacterial isolates showed negative results. Moreover, this method could be used as a better alternative to PCR based methods, for early detection of the pathogens.

scholarly journals Establishment and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pseudomonas fluorescens in Raw Milk

2022 ◽  
Vol 12 ◽  
Author(s):  
Yushan Bu ◽  
Wenjun Qiao ◽  
Zhengyuan Zhai ◽  
Tongjie Liu ◽  
Pimin Gong ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Rapid Detection ◽  
Detection Threshold ◽  
Lamp Assay ◽  
Raw Milk ◽  
Isothermal Amplification ◽  
Cold Chain ◽  
Protease Gene ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay

Raw milk is susceptible to microbial contamination during transportation and storage. Pseudomonas fluorescens producing heat-resistant enzymes have become the most common and harmful psychrophilic microorganisms in the cold chain logistics of raw milk. To rapidly detect P. fluorescens in raw milk, the protease gene aprX was selected as a detection target to construct a set of primers with strong specificity, and a loop-mediated isothermal amplification (LAMP) assay was established. The detection thresholds of the LAMP assay for pure cultured P. fluorescens and pasteurized milk were 2.57 × 102 and 3 × 102 CFU/mL, respectively. It had the advantages over conventional method of low detection threshold, strong specificity, rapid detection, and simple operation. This LAMP assay can be used for online monitoring and on-site detection of P. fluorescens in raw milk to guarantee the quality and safety of dairy products.

Rapid detection of Helicobacter pylori using cytotoxin-associated gene A based on loop-mediated isothermal amplification assay and magnetic nanoparticles

2020 ◽  
Vol 10 (2) ◽  
pp. 283-289
Author(s):  
Junwang Zhang ◽  
Meixia Wang ◽  
Ying Shi ◽  
Qi Wang ◽  
Wubo Zhao
Keyword(s):  
Helicobacter Pylori ◽  
Magnetic Nanoparticles ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Bacterial Strains ◽  
Loop Mediated Isothermal Amplification ◽  
Cytotoxin Associated Gene A ◽  
H Pylori ◽  
Sensitive Method

The current methods for detecting Helicobacter pylori infection are time-consuming and have relatively low sensitivity. More appropriate tests are needed. A rapid, specific, and sensitive method was presently developed to detect the cytotoxin-associated gene A (cagA) of H. pylori. Genomic DNA was extracted using magnetic nanoparticles and then amplified by the loop-mediated isothermal amplification (LAMP) reaction using primers we designed. To assess the diagnostic value of the LAMP assay in detecting H. pylori cagA, agarose gel electrophoresis as well as detection of fluorescence intensity after adding fluorescent dye were done. Specificity analysis showed that 11 pathogenic bacterial strains common in human gut were negative for cagA, with a positive result obtained only for H. pylori. Sensitivity analysis demonstrated a cagA detection limit of 100 fg. The results were consistent with that of the 3C-urea breath test. The novel LAMP assay can directly identify H. pylori cagA in the gastric juice of clinical patients with high sensitivity and specificity. The comparatively more rapid and more sensitive method may be valuable for clinical applications.

scholarly journals A Loop-Mediated Isothermal Amplification Assay Targeting Neisseria gonorrhoeae penA-60.001

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Ken Shimuta ◽  
Shu-ichi Nakayama ◽  
Hideyuki Takahashi ◽  
Makoto Ohnishi
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Assay System ◽  
Line Treatment ◽  
First Line ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
First Line Treatment

ABSTRACT Ceftriaxone (CRO) is widely used as the first-line treatment for gonococcal infections. However, CRO-resistant Neisseria gonorrhoeae strains carrying mosaic penA-60.001 have emerged recently and disseminated worldwide. To meet the urgent need to detect these strains, we report here a loop-mediated isothermal amplification (LAMP) assay system that targets N. gonorrhoeae penA-60.001. This assay system can differentiate N. gonorrhoeae strains carrying mosaic penA-60.001 from strains carrying other penA alleles.

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