A rapid, simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples

Okada, Kazuhisa; Chantaroj, Siriporn; Taniguchi, Tooru; Suzuki, Yasuhiko; Roobthaisong, Amonrattana; Puiprom, Orapim; Honda, Takeshi; Sawanpanyalert, Pathom

doi:10.1016/j.diagmicrobio.2009.09.004

Cited by 51 publications

(43 citation statements)

References 14 publications

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“…In general, molecular level-based detection methods such as PCR and LAMP are subjected to a variety of inhibitors present in clinical samples. Some researchers have reported that the Bst polymerase in LAMP is less sensitive to the presence of inhibitors than the Taq polymerase used in classic PCR [25], [26]. Our results showed that the LAMP assay is more accurate and sensitive than qPCR methods using simulated human stool samples, and proved markedly faster than qPCR by at least 20 min, thereby significantly shortening the total assay time.…”

Section: Discussionmentioning

confidence: 58%

Rapid and Sensitive Detection of Plesiomonas shigelloides by Loop-Mediated Isothermal Amplification of the hugA Gene

Shao¹,

Xu²,

Xiong³

et al. 2012

PLoS ONE

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Plesiomonas shigelloides is one of the causative agents of human gastroenteritis, with increasing number of reports describing such infections in recent years. In this study, the hugA gene was chosen as the target to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, and sensitive detection of P. shigelloides. The performance of the assay with reference plasmids and spiked human stools as samples was evaluated and compared with those of quantitative PCR (qPCR). No false-positive results were observed for the 32 non-P. shigelloides strains used to evaluate assay specificity. The limit of detection for P. shigelloides was approximately 20 copies per reaction in reference plasmids and 5×103 CFU per gram in spiked human stool, which were more sensitive than the results of qPCR. When applied in human stool samples spiked with 2 low levels of P. shigelloides, the LAMP assays achieved accurate detection after 6-h enrichment. In conclusion, the LAMP assay developed in this study is a valuable method for rapid, cost-effective, and simple detection of P. shigelloides in basic clinical and field laboratories in the rural areas of China.

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Section: Discussionmentioning

confidence: 58%

Rapid and Sensitive Detection of Plesiomonas shigelloides by Loop-Mediated Isothermal Amplification of the hugA Gene

Shao¹,

Xu²,

Xiong³

et al. 2012

PLoS ONE

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“…Of the 42 samples, 26 tested positive by LAMP assay, while only 1 and 3 samples tested positive by nested PCR and real-time PCR, respectively. Some researchers have reported that the Bst polymerase in LAMP is less sensitive to the presence of inhibitors than is the Taq polymerase used in PCR (8,16,13). Thus, although nested PCR (1, 9) and real-time PCR (5,18) have been reported to be more sensitive than the LAMP assay for the detection of A. phagocytophilum, in this study, the LAMP assay was shown to be more accurate and more sensitive than PCR methods on clinical samples.…”

Section: Discussionmentioning

confidence: 69%

“…Thus, we suggest that A. phagocytophilum infection When evaluating the specificity of the msp2 LAMP assay, we tested 27 members of the order Rickettsiales and other pathogenic bacteria, and the results of this study showed that the specificity of the LAMP assay is 100%. The high specificity of LAMP methods has also been reported for other pathogens (4,11,13,14,16). Compared to conventional PCR techniques, 6 LAMP primers were designed to target 8 regions, while PCR methods used primers targeting 2 to 4 regions.…”

Section: Discussionmentioning

confidence: 99%

Rapid, Simple, and Sensitive Detection of Anaplasma phagocytophilum by Loop-Mediated Isothermal Amplification of the msp2 Gene

et al. 2011

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Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis, which is prevalent throughout China. In this study, we describe a rapid, simple, and sensitive loop-mediated isothermal amplification (LAMP) assay targeting the msp2 gene of A. phagocytophilum that is ideal for application in rural areas in China. This assay has the potential to detect A. phagocytophilum early in infection as an alternative to existing methods. A total of 42 suspected cases of infection with A. phagocytophilum, 15 serologically confirmed and 27 probable cases, were analyzed by the msp2 LAMP assay. To validate the accuracy of LAMP, previously established nested-PCR and real-time PCR assays were utilized. The sensitivity of LAMP was 25 copies per reaction (approximately 1,250 copies per ml blood) for A. phagocytophilum, and the assay did not detect false positives among 27 members of the order Rickettsiales and 17 common clinical pathogens. To evaluate the clinical applicability of the LAMP assay, a total of 42 clinical samples were examined. A positive LAMP result was obtained for 12 of the confirmed cases and for 14 of 27 suspected cases, while only 1 confirmed case and 3 cases (2 confirmed cases and 1 suspected case) were detected by nested PCR and real-time PCR, respectively. The LAMP assay described in this study demonstrated a high level of sensitivity comparable with that of nested PCR and real-time PCR for the detection of A. phagocytophilum. This LAMP assay is a valuable method for rapid, cost-effective, and simple detection of A. phagocytophilum in the rural areas of China.Human granulocytic anaplasmosis (HGA) is an emerging tick-borne disease caused by Anaplasma phagocytophilum, which was reported in the United States in 1990 and in Europe in 1997 (3, 6). Seroepidemiological data suggest that HGA infection rates in the United States are as high as 15% to 36% in some locations (7). An unusual case of nosocomial humanto-human A. phagocytophilum transmission was reported in 2006 in a regional hospital in Anhui Province, China (19). Subsequently, nationwide seroepidemiological surveys of A. phagocytophilum among high-risk agricultural groups in China demonstrated that the total seroprevalence rate of A. phagocytophilum was 13.94% (unpublished data). However, the greatest challenge to clinicians is the lack of a rapid, early, and accurate diagnostic method for the detection of anaplasmosis as an emerging infectious disease in China. The indirect immunofluorescence assay (IFA), the gold standard method proposed by the World Health Organization Collaborating Center for Rickettsial Reference and Research, depends on a specific, expensive fluorescence microscope and requires a pair of serum samples from both the acute and convalescent stages of the illness, which is not applicable as an early test for A. phagocytophilum infection. The utility of insensitive conventional PCR, time-consuming and easily contaminated nested PCR, and highly sensitive real-time PCR is also limited in rural areas because they require ...

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“…Individual colonies were selected and cultured for 18 h at 37°C on Tryptic Soy Broth (TSB) (Difco, Detroit, MI). In addition, Loop-mediated isothermal amplification (LAMP) [25] was performed to screen for toxigenic V. cholerae among approximately 2,000 samples. Biotyping of the isolates was carried out using the Voges-Proskauer (VP) reaction and polymyxin B (50 units) [26].…”

Section: Methodsmentioning

confidence: 99%

Genotypic and PFGE/MLVA Analyses of Vibrio cholerae O1: Geographical Spread and Temporal Changes during the 2007–2010 Cholera Outbreaks in Thailand

Okada

Roobthaisong²,

Nakagawa

et al. 2012

PLoS ONE

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Background Vibrio cholerae O1 El Tor dominated the seventh cholera pandemic which occurred in the 1960s. For two decades, variants of V. cholerae O1 El Tor that produce classical cholera toxin have emerged and spread globally, replacing the prototypic El Tor biotype. This study aims to characterize V. cholerae O1 isolates from outbreaks in Thailand with special reference to genotypic variations over time.Methods/FindingsA total of 343 isolates of V. cholerae O1 from cholera outbreaks from 2007 to 2010 were investigated, and 99.4% were found to carry the classical cholera toxin B subunit (ctxB) and El Tor rstR genes. Pulsed-field gel electrophoresis (PFGE) differentiated the isolates into 10 distinct pulsotypes, clustered into two major groups, A and B, with an overall similarity of 88%. Ribotyping, multiple-locus variable-number tandem-repeat analysis (MLVA), and PCR to detect Vibrio seventh pandemic island II (VSP-II) related genes of randomly selected isolates from each pulsotype corresponded to the results obtained by PFGE. Epidemiological investigations revealed that MLVA type 2 was strongly associated with a cholera outbreak in northeastern Thailand in 2007, while MLVA type 7 dominated the outbreaks of the southern Gulf areas in 2009 and MLVA type 4 dominated the outbreaks of the central Gulf areas during 2009–2010. Only MLVA type 16 isolates were found in a Thai-Myanmar border area in 2010, whereas those of MLVA types 26, 39, and 41 predominated this border area in 2008. Type 39 then disappeared 1–2 years later as MLVA type 41 became prevalent. Type 41 was also found to infect an outbreak area.ConclusionsMLVA provided a high-throughput genetic typing tool for understanding the in-depth epidemiology of cholera outbreaks. Our epidemiological surveys suggest that some clones of V. cholerae O1 with similar but distinctive genetic traits circulate in outbreak sites, while others disappear over time.

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A rapid, simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples

Cited by 51 publications

References 14 publications

Rapid and Sensitive Detection of Plesiomonas shigelloides by Loop-Mediated Isothermal Amplification of the hugA Gene

Rapid and Sensitive Detection of Plesiomonas shigelloides by Loop-Mediated Isothermal Amplification of the hugA Gene

Rapid, Simple, and Sensitive Detection of Anaplasma phagocytophilum by Loop-Mediated Isothermal Amplification of the msp2 Gene

Genotypic and PFGE/MLVA Analyses of Vibrio cholerae O1: Geographical Spread and Temporal Changes during the 2007–2010 Cholera Outbreaks in Thailand

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