Dataset Information

Rapid and sensitive detection of Listeria ivanovii by loop-mediated isothermal amplification of the smcL gene.

ABSTRACT: A loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six primers targeting eight regions on the species-specific gene smcL. The LAMP assay could be completed within 1 h at 64°C in a water bath. Amplification products were directly observed by the Loopamp Fluorescent Detection Reagent (FD) or detected by agarose gel electrophoresis. Moreover, the LAMP reactions were also detected by real-time measurement of turbidity. The exclusivity of 77 non-L. ivanovii and the inclusivity of 17 L. ivanovii were both 100% in the assay. Sensitivity of the LAMP assay was 250 fg DNA and 16 CFU per reaction for detection of L. ivanovii in pure cultures and simulated human stool. The LAMP assay was 10 and 100-fold more sensitive than quantitative PCR (qPCR) and conventional PCR assays,respectively. When applied to human stool samples spiked with low level (8 CFU/0.5 g) of L. ivanovii strains, the new LAMP assay described here achieved positive detection after 6 hours enrichment. In conclusion, the new LAMP assay in this study can be used as a valuable, rapid and sensitive detection tool for the detection of L. ivanovii in field, medical and veterinary laboratories.

SUBMITTER: Wang Y

PROVIDER: S-EPMC4280119 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Rapid and sensitive detection of Listeria ivanovii by loop-mediated isothermal amplification of the smcL gene.

Wang Yi Y Wang Yan Y Xu Huaqing H Dai Hang H Meng Shuang S Ye Changyun C

PloS one 20141230 12

A loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six primers targeting eight regions on the species-specific gene smcL. The LAMP assay could be completed within 1 h at 64°C in a water bath. Amplification products were directly observed by the Loopamp F ...[more]

PMID: 25549337

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Project description:BackgroundQ fever is the most widespread zoonosis, and domestic animals are the most common sources of transmission. It is not only difficult to distinguish from other febrile diseases because of the lack of specific clinical manifestations in humans, but it is also difficult to identify the disease in C. burnetii-carrying animals because of the lack of identifiable features. Conventional serodiagnosis requires sera from the acute and convalescent stages of infection, which are unavailable at early diagnosis. Nested PCR and real-time PCR require equipment. In this study, we developed a Loop-Mediated Isothermal Amplification (LAMP) assay to identify C. burnetii rapidly and sensitively.MethodsA universal LAMP primer set was designed to detect the repeated sequence IS1111a of the htpAB gene of C. burnetii using PrimerExplorer V4 software. The sensitivity of the LAMP assay was evaluated using known quantities of recombined reference plasmids containing the targeted genes. The specificity of the developed LAMP assay was determined using 26 members of order Rickettsiae and 18 other common pathogens. The utility of the LAMP assay was further compared with real time PCR by the examination 24 blood samples including 6 confirmed and 18 probable Q fever cases, which diagnosed by IFA serological assessment and real time PCR. In addition, 126 animal samples from 4 provinces including 97 goats, 7 cattle, 18 horses, 3 marmots and 1 deer were compared by these two methods.ResultsThe limits of detection of the LAMP assay for the htpAB gene were 1 copy per reaction. The specificity of the LAMP assay was 100%, and no cross-reaction was observed among the bacteria used in the study. The positive rate of unknown febrile patients was 33.3%(95%CI 30.2%-36.4%) for the LAMP assay and 8.3%(95%CI 7.4%-9.2%) for the real time PCR(P<0.05). Similarly, the total positive rate of animals was 7.9%(95%CI 7.1%-8.7%) for the LAMP assay and 0.8%(95%CI 0.7%-0.9%)for the real time PCR(P<0.01). Using the developed LAMP assay, Q fever in the Yi Li area, Xinjiang Province, was confirmed.ConclusionsThe LAMP assay is a potential tool to support the diagnosis of Q fever in humans and domestic animals in the field, especially in the rural areas of China, because of its rapid and sensitive detection without the aid of sophisticated equipment or a complicated protocol.

Dataset Information

Rapid and sensitive detection of Listeria ivanovii by loop-mediated isothermal amplification of the smcL gene.

Publications

Rapid and sensitive detection of Listeria ivanovii by loop-mediated isothermal amplification of the smcL gene.

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