scholarly journals Direct detection of Mycobacterium avium in environmental water and scale samples by loop-mediated isothermal amplification

2013 ◽  
Vol 12 (2) ◽  
pp. 211-219 ◽  
Author(s):  
Yukiko Nishiuchi ◽  
Aki Tamaru ◽  
Yasuhiko Suzuki ◽  
Seigo Kitada ◽  
Ryoji Maekura ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Environmental Samples ◽  
Direct Detection ◽  
Culture Method ◽  
Environmental Water ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Culture Methods ◽  
Loop Mediated Isothermal Amplification ◽  
Conventional Culture

We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA/reaction for M. avium. Using an FTA® elute card, DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment.

Rapid Detection of Mycoplasma pneumoniae by Loop-Mediated Isothermal Amplification (LAMP) in Clinical Respiratory Specimens

2019 ◽  
Author(s):  
Maryam ARFAATABAR ◽  
Narjes NOORI GOODARZI ◽  
Davoud AFSHAR ◽  
Hamed MEMARIANI ◽  
Ghasem AZIMI ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Culture Method ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Culture Methods ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Respiratory Specimens

  Background: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP) worldwide, especially among children and debilitated populations. The present study aimed to investigate a loop-mediated isothermal amplification (LAMP) technique for rapid detection of M. pneumoniae in clini-cal specimens collected from patients with pneumonia. Methods: Throat swabs were collected from 110 outpatients who suffered from pneumonia. Throat swab samples were obtained from patients referred to the hospital outpatient clinics of Tehran University hospitals, Iran in 2017. The presence of M. pneumoniae in the clinical specimens was evaluated by LAMP, PCR and culture methods. Sensitivity and specificity of the LAMP and PCR assays were also determined. Results: Out of 110 specimens, LAMP assay detected M. pneumoniae in 35 specimens. Detection limit of the LAMP assay was determined to be 33fg /μL or ~ 40 genome copies/reaction. Moreover, no cross-reaction with genomic DNA from other bacteria was observed. Only 25 specimens were positive by the culture method. The congruence between LAMP assay and culture method was ‘substantial’ (κ=0.77). Specificity and sensitivity of LAMP assay were 88.2%, 100% in compare with culture. However, the con-gruence between LAMP assay and PCR assay was ‘almost perfect’ (κ=0.86). Specificity and sensitivity of LAMP assay were 92.5%, 100% in compare with PCR. Conclusion: Overall, the LAMP assay is a rapid and cost-efficient laboratory test in comparison to other methods including PCR and culture. Therefore, the LAMP method can be applied in identification of M. pneumoniae isolates in respiratory specimens.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of viable but non-culturable Vibrio cholerae O1

2021 ◽  
Author(s):  
Parastoo Chamanrokh ◽  
Rita R. Colwell ◽  
Anwar Huq
Keyword(s):  
Vibrio Cholerae ◽  
Culture Media ◽  
Lamp Assay ◽  
Environmental Water ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Waterborne Pathogen ◽  
Loop Mediated Isothermal Amplification ◽  
Added Benefit ◽  
Polymerase Chain

<i>Vibrio cholerae</i>, an important waterborne pathogen, is a rod-shaped bacterium that naturally exists in aquatic environments. When conditions are unfavorable for growth, the bacterium can undergo morphological and physiological changes to assume a coccoid morphology. This stage in its life cycle is referred to as viable but non-culturable (VBNC) since VBNC cells do not grow on conventional bacteriological culture media. The current study compared polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to detect and identify VBNC <i>V. cholerae</i>. Because it is difficult to detect and identify VBNC <i>V. cholerae</i>, the results of the current study are useful in showing LAMP to be more sensitive and rapid than PCR in detecting and identifying non-culturable, coccoid forms of <i>V. cholerae</i>. Furthermore, the LAMP method is effective in detecting and identifying very low numbers of coccoid VBNC <i>V. cholerae</i> in environmental water samples, with the added benefit of being inexpensive to perform.

scholarly journals Elevated OPN, IP-10, and Neutrophilia in Loop-Mediated Isothermal Amplification Confirmed Tuberculosis Patients

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Beata Shiratori ◽  
Susan Leano ◽  
Chie Nakajima ◽  
Haorile Chagan-Yasutan ◽  
Toshiro Niki ◽  
...  
Keyword(s):  
Leukocyte Count ◽  
Acute Stage ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Culture Methods ◽  
Tuberculosis Patients ◽  
Loop Mediated Isothermal Amplification ◽  
Metro Manila ◽  
Healthy Control ◽  
Proper Diagnosis

Tuberculosis (TB) is the second most common cause of death from infectious diseases and results in high socioeconomic losses to many countries. Proper diagnosis is the first step in TB eradication. To develop a rapid, simple, and accurate diagnostic TB test and to characterize the prevalence ofMycobacterium tuberculosis(MTB) genotypes and immune profiles of TB patients, a total of 37 TB patients and 30 healthy control (HC) from Metro Manila were enrolled. Loop-mediated isothermal amplification (LAMP) reliably detected MTB infection. Manila genotype was identified by spoligotyping method in all TB patients. Osteopontin (OPN), interferon-γ-induced protein 10 kDa (IP-10), and neutrophil counts were found to reflect the acute stage of MTB infection. The sensitivity and specificity were 94.6% and 93.3%, respectively, for both OPN and IP-10, and they were 83.8% and 78.6%, respectively, for neutrophils. The combination of OPN, IP-10, neutrophil count, IL-6, IL-8, TNF-α, MCP-1, platelets, galectin-9, and leukocyte count correctly identifies all the HC and 96.3% of TB patients. LAMP method may serve as a rapid, supportive method in addition to time-consuming culture methods. OPN, IP-10, and neutrophil counts are useful in detecting MTB infection and may have utility in monitoring the course of the disease.

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid and Sensitive Detection of Vibrio parahaemolyticus

2011 ◽  
Vol 74 (9) ◽  
pp. 1462-1467 ◽  
Author(s):  
JIRO NEMOTO ◽  
MASANARI IKEDO ◽  
TADASHI KOJIMA ◽  
TAKAYOSHI MOMODA ◽  
HIROTAKA KONUMA ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Lamp Assay ◽  
Culture Method ◽  
Isothermal Amplification ◽  
Most Probable Number ◽  
Lamp Method ◽  
Probable Number ◽  
Loop Mediated Isothermal Amplification ◽  
Negative Results ◽  
Rpod Gene

Loop-mediated isothermal amplification (LAMP) assays targeting the rpoD and toxR genes were developed to detect Vibrio parahaemolyticus. All 78 tested V. parahaemolyticus strains yielded positive results within 40 min, while negative results were obtained for 69 strains of other organisms even at 60 min. For V. parahaemolyticus ATCC 17802 in pure culture, the detection limits of LAMP assays targeting rpoD and toxR were 3.7 and 450 CFU per test, respectively. Due to the higher sensitivity of rpoD-LAMP, it was further evaluated for the ability to detect V. parahaemolyticus in seafood samples. V. parahaemolyticus populations spiked in short-necked clams were enumerated by the most-probable-number (MPN) method combined with the rpoD-LAMP assay and the MPN method with a culture method using agar medium. The MPN-rpoD-LAMP method had better sensitivity and was more rapid than the conventional method. These results indicate that the MPN-LAMP assay targeting the rpoD gene is a specific, sensitive, and rapid method to enumerate V. parahaemolyticus organisms.

scholarly journals Comparison of Loop-Mediated Isothermal Amplification Assay and Conventional Culture Methods for Detection of Campylobacter jejuni and Campylobacter coli in Naturally Contaminated Chicken Meat Samples

2009 ◽  
Vol 75 (6) ◽  
pp. 1597-1603 ◽  
Author(s):  
Wataru Yamazaki ◽  
Masumi Taguchi ◽  
Takao Kawai ◽  
Kentaro Kawatsu ◽  
Junko Sakata ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Lamp Assay ◽  
Chicken Meat ◽  
Isothermal Amplification ◽  
Campylobacter Coli ◽  
Culture Methods ◽  
Culture Test ◽  
Loop Mediated Isothermal Amplification ◽  
Conventional Culture ◽  
Meat Samples

ABSTRACT We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for detection of chicken meat samples naturally contaminated with Campylobacter jejuni and Campylobacter coli. A total of 144 Preston enrichment broth cultures from chicken meat samples were assessed by using the LAMP assay and conventional culture methods, which consist of a combination of Preston enrichment culturing and plating onto Butzler and modified charcoal cefoperazone deoxycholate agars. Compared with C. jejuni-C. coli isolation using the conventional culture test, the LAMP results showed 98.5% (67/68) and 97.4% (74/76) sensitivity and specificity, respectively, and the positive and negative predictive values were 97.1% (67/69) and 98.7% (74/75), respectively. The conventional culture test required more than 3 to 4 days to isolate and identify C. jejuni and C. coli in the Preston enrichment cultures. In contrast, the LAMP assay was markedly faster, requiring less than 90 min from the beginning of DNA extraction to final detection and differentiation of C. jejuni and C. coli. In total, the LAMP assay required 23.5 to 25.5 h from the beginning of the enrichment culture to final determination. These results suggest that our LAMP assay is a powerful tool for rapid, sensitive, and practical detection of C. jejuni and C. coli which may facilitate surveillance and control of C. jejuni-C. coli contamination in chicken, as well as investigations of food poisoning incidents caused by these organisms. This is the first report of a highly sensitive and specific LAMP assay to detect and differentiate C. jejuni and C. coli in chicken meat samples.

scholarly journals Efficient and Specific Detection ofSalmonellain Food Samples Using astn-Based Loop-Mediated Isothermal Amplification Method

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Mevaree Srisawat ◽  
Watanalai Panbangred
Keyword(s):  
Lamp Assay ◽  
High Sensitivity ◽  
High Specificity ◽  
Culture Method ◽  
Food Samples ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Method ◽  
Pure Cultures

TheSalmonellaenterotoxin (stn) gene exhibits high homology amongS. entericaserovars andS. bongori. A set of 6 specific primers targeting thestngene were designed for detection ofSalmonellaspp. using the loop-mediated isothermal amplification (LAMP) method. The primers amplified target sequences in all 102 strains of 87 serovars ofSalmonellatested and no products were detected in 57 non-Salmonellastrains. The detection limit in pure cultures was 5 fg DNA/reaction when amplified at 65°C for 25 min. The LAMP assay could detectSalmonellain artificially contaminated food samples as low as 220 cells/g of food without a preenrichment step. However, the sensitivity was increased 100-fold (~2 cells/g) following 5 hr preenrichment at 35°C. The LAMP technique, with a preenrichment step for 5 and 16 hr, was shown to give 100% specificity with food samples compared to the reference culture method in which 67 out of 90 food samples gave positive results. Different food matrixes did not interfere with LAMP detection which employed a simple boiling method for DNA template preparation. The results indicate that the LAMP method, targeting thestngene, has great potential for detection ofSalmonellain food samples with both high specificity and high sensitivity.

scholarly journals Isolation of Mycobacterium avium Subsp. Paratuberculosis in the Feces and Tissue of Small Ruminants Using a Non-Automated Liquid Culture Method

Animals ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 20
Author(s):  
Luigi De Grossi ◽  
Davide Santori ◽  
Antonino Barone ◽  
Silvia Abbruzzese ◽  
Matteo Ricchi ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Liquid Culture ◽  
Culture Media ◽  
Small Ruminants ◽  
Culture Method ◽  
Mycobacterium Avium Subsp ◽  
Type I ◽  
Culture Methods ◽  
Specific Pcr ◽  
Mycobacterium Avium Subsp Paratuberculosis

Paratuberculosis is a chronic disease of ruminants caused by Mycobacterium avium subsp. Paratuberculosis (MAP). Since isolation of MAP type I (S) is rarely reported in Italy, our research was aimed at isolating, by an inexpensive liquid culture manual method, this type of MAP isolates. At first, we used an ELISA to point out to serologically positive samples from five flocks. Secondly, we used a fecal direct IS900-qPCR on the ELISA positive samples, in order to detect shedder animals. Feces from IS900-qPCR positive samples were inoculated in solid and liquid culture media. IS900-qPCR was further used to test the growth of MAP isolates in liquid medium, which were further confirmed by f57-qPCR and submitted to typing by specific PCR in order to identify the MAP type. Twenty-eight samples (24 fecal and four tissutal samples) were processed by culture methods, resulting in the isolation of six type I MAP field isolates. Notably, no isolates were recovered by solid media, underlining the utility of this liquid method. Few data about this type of MAP are currently available in Italy, and further analyses should be carried out in order to study the origin and epidemiology of type I strains circulating in Italy.

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