Comparison of the Loop-Mediated Isothermal Amplification (LAMP) Method and Conventional Culture Method for the Detection of Campylobacter Species from Retail Chickens

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.

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

Iranian Journal of Public Health ◽

10.18502/ijph.v48i5.1809 ◽

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.

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

Journal of Food Protection ◽

10.4315/0362-028x.jfp-10-519 ◽

2011 ◽

Vol 74 (9) ◽

pp. 1462-1467 ◽

Cited By ~ 21

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.

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

BioMed Research International ◽

10.1155/2015/356401 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 10

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.

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Prevalence and Antimicrobial Susceptibility of Salmonella Isolated from a Variety of Raw Meat Sausages in Gaborone (Botswana) Retail Stores

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-438 ◽

2012 ◽

Vol 75 (4) ◽

pp. 637-642 ◽

Cited By ~ 4

Author(s):

RONALD GAELEKOLWE SAMAXA ◽

MAITSHWARELO IGNATIUS MATSHEKA ◽

SUNUNGUKO WATA MPOLOKA ◽

BERHANU ABEGAZ GASHE

Keyword(s):

Antimicrobial Susceptibility ◽

Salmonella Enterica ◽

Antimicrobial Agents ◽

Culture Method ◽

Multidrug Resistant ◽

Pcr Assay ◽

Raw Meat ◽

Conventional Culture ◽

The Difference ◽

Conventional Culture Method

The objective of the study was to provide baseline data on the prevalence and antimicrobial susceptibility of Salmonella in different types of raw meat sausages directly accessible to the consumers in Gaborone, Botswana. A total of 300 raw sausages comprising 79 beef, 78 pork, 72 chicken, and 71 mutton samples were concurrently analyzed for the presence of Salmonella using a conventional culture method and a validated PCR method. The PCR assay results were in full concordance with those of the conventional culture method for the detection of Salmonella. Sixty-five (21.7%) of 300 samples were positive for Salmonella by both the conventional culture method and PCR assay. Even though more chicken samples contained Salmonella than did any other sausage type, the difference in the presence of Salmonella among the four sausages types was not significant. Eleven serotypes were identified, and Salmonella enterica subsp. salamae II was most prevalent in all the sausage types. Beef sausages generally had higher mesophilic bacterial counts than did the other three sausage types. However, higher microbial counts were not reflective of the presence of salmonellae. Susceptibility of the Salmonella enterica serotypes to 20 antimicrobial agents was determined, and Salmonella Muenchen was resistant to the widest array of agents and was mostly isolated from chicken sausages. Regardless of the meat of origin, all 65 Salmonella isolates were resistant to at least four antimicrobial agents: amikacin, gentamicin, cefuroxime, and tombramycin. This resistance profile group was the most common in all four sausage types, comprising 90% of all Salmonella isolates from beef, 71% from pork, 63% from mutton, and 35% from chicken. These results suggest that raw sausages pose a risk of transmitting multidrug-resistant Salmonella isolates to consumers.

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Rapid and sensitive diagnosis of adenoviral keratoconjunctivitis by loop-mediated isothermal amplification (LAMP) method

Current Eye Research ◽

10.1080/02713680490503796 ◽

2004 ◽

Vol 28 (6) ◽

pp. 445-450 ◽

Cited By ~ 8

Author(s):

Taketoshi Wakabayashi ◽

Ryoko Yamashita ◽

Tetsuhiko Kakita ◽

Mito Kakita ◽

Tetsuro Oshika

Keyword(s):

Isothermal Amplification ◽

Lamp Method ◽

Loop Mediated Isothermal Amplification

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Application of loop mediated isothermal amplification (LAMP) method for detection of Edwarsiella ictaluri on patin (Pangasius sp.) fish

IOP Conference Series Earth and Environmental Science ◽

10.1088/1755-1315/744/1/012050 ◽

2021 ◽

Vol 744 (1) ◽

pp. 012050

Author(s):

H Novita ◽

U Purwaningsih ◽

A M Lusiastuti

Keyword(s):

Isothermal Amplification ◽

Lamp Method ◽

Loop Mediated Isothermal Amplification

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High Prevalence of yadA-Positive Yersinia enterocolitica in Pig Tongues and Minced Meat at the Retail Level in Finland

Journal of Food Protection ◽

10.4315/0362-028x-62.2.123 ◽

1999 ◽

Vol 62 (2) ◽

pp. 123-127 ◽

Cited By ~ 56

Author(s):

MARIA FREDRIKSSON-AHOMAA ◽

SEBASTIAN HIELM ◽

HANNU KORKEALA

Keyword(s):

Yersinia Enterocolitica ◽

Culture Method ◽

Selective Enrichment ◽

Conventional Culture ◽

High Prevalence ◽

Retail Outlets ◽

Polymerase Chain ◽

Meat Samples ◽

Minced Meat ◽

Conventional Culture Method

Polymerase chain reaction (PCR) was used to determine the prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland and to confirm the yadA-positive Y. enterocolitica isolates recovered from the same samples using the conventional culture method. A total of 51 pig tongues purchased at 12 retail outlets and 255 minced meat samples purchased at 40 retail outlets in the Helsinki area were studied. The prevalence of Y. enterocolitica carrying the yadA gene was 92% in pig tongues and 25% in minced meat using PCR and 78% in tongues and 2% in minced meat with the culture method. The prevalence of yadA-positive tongues was higher (98%) when both PCR- and culture-positive results were included because Y. enterocolitica carrying the yadA gene could also be isolated in three PCR-negative tongue samples. In the minced meat samples, all PCR-negative samples were also culture-negative. With the culture method, 66 of 80 yadA-positive isolates in 38 tongues and all yadA-positive isolates (4) in four minced meat samples were recovered after selective enrichment. A total of 92 isolates of Y. enterocolitica bioserotype 4/O:3 in tongues and 5 isolates in minced meat were found, of which 13% in tongues and 20% in minced meat did not carry the yadA gene.

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Loop-Mediated Isothermal Amplification for Detection ofStaphylococcus aureusin Dairy Cow Suffering from Mastitis

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/435982 ◽

2012 ◽

Vol 2012 ◽

pp. 1-5 ◽

Cited By ~ 9

Author(s):

Zhang Tie ◽

Wang Chunguang ◽

Wei Xiaoyuan ◽

Zhao Xinghua ◽

Zhong Xiuhui

Keyword(s):

Staphylococcus Aureus ◽

High Sensitivity ◽

High Specificity ◽

Isothermal Amplification ◽

Lamp Method ◽

Specific Primers ◽

Loop Mediated Isothermal Amplification ◽

Reaction Tube ◽

Specificity And Sensitivity ◽

Detectable Concentration

To develop a rapid detection method ofStaphylococcus aureususing loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of thenucgene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was1×102 CFU/mL and that of PCR was1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection ofStaphylococcus aureushas many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection ofStaphylococcus aureus.

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