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 Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli

2008 ◽  
Vol 57 (4) ◽  
pp. 444-451 ◽  
Author(s):  
Wataru Yamazaki ◽  
Masumi Taguchi ◽  
Masanori Ishibashi ◽  
Miyoshi Kitazato ◽  
Masafumi Nukina ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Lamp Assay ◽  
Test Tube ◽  
Isothermal Amplification ◽  
Campylobacter Coli ◽  
Lamp Product ◽  
Direct Plating ◽  
Loop Mediated Isothermal Amplification ◽  
Simple Detection ◽  
Stool Specimens

We developed a loop-mediated isothermal amplification (LAMP) assay for the rapid and simple detection of Campylobacter jejuni and Campylobacter coli. The assay provides a specific LAMP product for each of these two species. The assay correctly identified 65 C. jejuni and 45 C. coli strains, but not 75 non-C. jejuni/coli strains. The sensitivity of the LAMP assay for C. jejuni and C. coli in spiked human stool specimens was 5.6×103 c.f.u. g−1 (1.4 c.f.u. per test tube) and 4.8×103 c.f.u. g−1 (1.2 c.f.u. per test tube), respectively. When 90 stool specimens from patients with diarrhoea were tested by LAMP and direct plating, the LAMP results showed 81.3 % sensitivity and 96.6 % specificity compared to isolation of C. jejuni and C. coli by direct plating. Further, the LAMP assay required less than 2 h for detection of C. jejuni and C. coli in stool specimens. This LAMP assay is a rapid and simple tool for the detection of C. jejuni and C. coli and will be useful in facilitating the early diagnosis of food poisoning incidents caused by these organisms.

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.

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 simultaneous detection of Campylobacter spp. and Salmonella spp. in chicken samples by duplex loop-mediated isothermal amplification coupled with a lateral flow biosensor assay

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254029
Author(s):  
Thanawat Sridapan ◽  
Wanida Tangkawsakul ◽  
Tavan Janvilisri ◽  
Taradon Luangtongkum ◽  
Wansika Kiatpathomchai ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Chicken Meat ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Salmonella Spp ◽  
Promising Alternative ◽  
Loop Mediated Isothermal Amplification ◽  
Alternative Approach ◽  
Meat Samples ◽  
Sensitivity Specificity

Development of a simple, rapid and specific assay for the simultaneous detection of Campylobacter spp. and Salmonella spp. based on duplex loop-mediated isothermal amplification (d-LAMP), combined with lateral-flow biosensor (LFB) is reported herein. LAMP amplicons of both pathogens were simultaneously amplified and specifically differentiated by LFB. The specificity of the d-LAMP-LFB was evaluated using a set of 68 target and 12 non-target strains, showing 100% inclusivity and exclusivity. The assay can simultaneously detect Campylobacter and Salmonella strains as low as 1 ng and 100 pg genomic DNA per reaction, respectively. The lowest inoculated detection limits for Campylobacter and Salmonella species in artificially contaminated chicken meat samples were 103 CFU and 1 CFU per 25 grams, respectively, after enrichment for 24 h. Furthermore, compared to culture-based methods using field chicken meat samples, the sensitivity, specificity and accuracy of d-LAMP- LFB were 95.6% (95% CI, 78.0%-99.8%), 71.4% (95% CI, 29.0%-96.3%) and 90.0% (95% CI, 73.4%-97.8%), respectively. The developed d-LAMP-LFB assay herein shows great potentials for the simultaneous detection of the Campylobacter and Salmonella spp. and poses a promising alternative approach for detection of both pathogens with applications in food products.

Improving the Detection Accuracy and Time for Campylobacter jejuni and Campylobacter coli in Naturally Infected Live and Slaughtered Chicken Broilers Using a Real-Time Fluorescent Loop-Mediated Isothermal Amplification Approach

2019 ◽  
Vol 82 (2) ◽  
pp. 189-193 ◽  
Author(s):  
ISLAM IBRAHIM SABIKE ◽  
WATARU YAMAZAKI
Keyword(s):  
Supply Chain ◽  
Campylobacter Jejuni ◽  
Food Supply ◽  
Direct Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Diagnostic Sensitivity ◽  
Campylobacter Coli ◽  
Fluorescent Lamp ◽  
Food Supply Chain

ABSTRACT Rapid and accurate identification of Campylobacter-positive broiler flocks and carcasses expedites separation and control interventions before release into the food supply chain and directly facilitates a reduction in the prevalence of human campylobacteriosis. In this study, the diagnostic performance of fluorescent loop-mediated isothermal amplification (LAMP) assays for the direct detection of Campylobacter jejuni and Campylobacter coli in broiler cloacal and cecal samples were evaluated and compared with that of turbidimetric LAMP approaches investigated previously. The duplex fluorescent LAMP assay had significantly higher (P < 0.05) diagnostic sensitivity (93.1%, 54 of 58 samples) than did the turbidimetric LAMP assay (82.8%, 48 of 58 samples) for detecting C. jejuni and C. coli in broiler cloacal samples, whereas the singleplex fluorescent LAMP assay had equivalent diagnostic sensitivity. For cecal samples, the diagnostic sensitivity of the fluorescent LAMP assay (100%, 38 of 38 samples) was the same as that of the turbidimetric LAMP. Fluorescent LAMP significantly reduced (P < 0.05) the maximum detection time for Campylobacter-positive cloacal and cecal samples to 28 and 11 min, respectively, and reduced the influence of amplification inhibitors responsible for most false-negative results obtained for cloacal samples with the turbidimetric LAMP assay. The diagnostic accuracy of the fluorescent LAMP assay for the direct detection of C. jejuni and C. coli in cloacal and cecal samples was 97.7 and 100%, respectively. These findings indicate that fluorescent LAMP assays are robust, highly accurate, and field-applicable methods for the identification of C. jejuni and C. coli, which will allow more accurate monitoring of food safety at various stages of the food supply chain at farms and slaughter facilities.

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