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

Shao, Meng; Xu, Jianguo; Xiong, Yongqiang; Ye, Changyun

doi:10.1371/journal.pone.0041978

Cited by 17 publications

(21 citation statements)

References 26 publications

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“…This value is similar to those reported in previous studies, which detected 1, 20, 7.9, and 3.8 CFU per reaction for Escherichia coli O157:H7, Plesiomonas shigelloides, C. jejuni, and C. coli, respectively (30,31,40). The sensitivity of the LAMP method developed in this study was 10-to 1,000-fold higher than that of conventional PCR or multiplex PCR (30,40). In concordance with the results of previous studies, LAMP showed 100-fold-greater sensitivity than conventional PCR or multiplex PCR.…”

Section: Discussionsupporting

confidence: 92%

Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

et al. 2014

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This study aimed to develop a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Arcobacter species. Specific primers targeting the 23S ribosomal RNA gene were used to detect Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. The specificity of the LAMP primer set was assessed using DNA samples from a panel of Arcobacter and Campylobacter species, and the sensitivity was determined using serial dilutions of Arcobacter species cultures. LAMP showed a 10-to 1,000-fold-higher sensitivity than multiplex PCR, with a detection limit of 2 to 20 CFU per reaction in vitro. Whereas multiplex PCR showed cross-reactivity with Campylobacter species, the LAMP method developed in this study was more sensitive and reliable than conventional PCR or multiplex PCR for the detection of Arcobacter species.

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Section: Discussionsupporting

confidence: 92%

Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

et al. 2014

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“…A range of PCR assays targeting different genomic fragments have been developed for the detection of P. shigelloides (Table 9) (42,51,221,222,223,224,225). One of the earliest assays was configured by González-Rey et al (42) and targeted a specific variable region of the 23S rRNA gene previously identified by Van Camp et al (226).…”

Section: Laboratory Identification Isolation and Identificationmentioning

confidence: 99%

“…Primers targeting the hugA gene were shown to have high specificity for P. shigelloides when tested with a range of Gramnegative strains of different genera and were used by these workers to detect the organism in saltwater fish. Meng et al (225) developed a detection assay for P. shigelloides targeting hugA ( Table 9) that is based on a loop-mediated isothermal amplification (LAMP) reaction mediated by Bst DNA polymerase (227). When used to test spiked human stool samples, the assay proved to be more accurate, sensitive, and faster (in assay time) than quantitative PCR.…”

Section: Laboratory Identification Isolation and Identificationmentioning

confidence: 99%

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Plesiomonas shigelloides Revisited

Janda¹,

2016

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SUMMARYAfter many years in the familyVibrionaceae, the genusPlesiomonas, represented by a single species,P. shigelloides, currently resides in the familyEnterobacteriaceae, although its most appropriate phylogenetic position may yet to be determined. Common environmental reservoirs for plesiomonads include freshwater ecosystems and estuaries and inhabitants of these aquatic environs. Long suspected as being an etiologic agent of bacterial gastroenteritis, convincing evidence supporting this conclusion has accumulated over the past 2 decades in the form of a series of foodborne outbreaks solely or partially attributable toP. shigelloides. The prevalence ofP. shigelloidesenteritis varies considerably, with higher rates reported from Southeast Asia and Africa and lower numbers from North America and Europe. Reasons for these differences may include hygiene conditions, dietary habits, regional occupations, or other unknown factors. Other human illnesses caused byP. shigelloidesinclude septicemia and central nervous system disease, eye infections, and a variety of miscellaneous ailments. For years, recognizable virulence factors potentially associated withP. shigelloidespathogenicity were lacking; however, several good candidates now have been reported, including a cytotoxic hemolysin, iron acquisition systems, and lipopolysaccharide. WhileP. shigelloidesis easy to identify biochemically, it is often overlooked in stool samples due to its smaller colony size or relatively low prevalence in gastrointestinal samples. However, one FDA-approved PCR-based culture-independent diagnostic test system to detect multiple enteropathogens (FilmArray) includesP. shigelloideson its panel. Plesiomonads produce β-lactamases but are typically susceptible to many first-line antimicrobial agents, including quinolones and carbapenems.

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“…The use of 6 oligonucleotides promotes the speed of the amplification and the specificity, and the output can be up to 10 9 copies of target DNA molecules within 1 h. The products of LAMP can be visualized in different ways, such as turbidity caused by the precipitation of magnesium pyrophosphate, gel electrophoresis, and the addition of complex ometric dyes, such as SYBR green to observe fluorescence under UV light. Recent reports showed that LAMP had been widely used in the detection of viruses and bacteria (16)(17)(18), but it has only recently been applied to some species in the filamentous fungi and yeast genera, including Aspergillus flavus, Aspergillus parasiticus, Aspergillus nomius, and Histoplasma capsulatum but not A. fumigatus as yet (19,20).…”

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confidence: 99%

Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus

et al. 2016

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Aspergillus fumigatus is a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection of A. fumigatus infection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection of A. fumigatus. The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatus strains, including 5 species of the Aspergillus genus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 10 2 copies for the same target. Clinical samples from a total of 69 patients with probable IA (n ‫؍‬ 14) and possible IA (n ‫؍‬ 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection of A. fumigatus in clinical testing has been developed.A spergillus fumigatus, a ubiquitous saprophytic fungus in most parts of the world, is a conditional pathogenic fungus and the major cause of invasive aspergillosis (IA). The infection is initiated by inhalation of conidia, which are cleared quickly in a normal host but can cause invasive disease in immunocompromised individuals (1). Despite many efforts during the last decade, the morbidity and mortality due to IA has been increasing (2). The poor therapeutic outcomes are associated with the weak host immune status and, importantly, the delayed detection and identification of the causative agent (3).The traditional culturing method, which is the gold standard for the diagnosis of A. fumigatus, requires several days for observation of growth, is time-consuming, and has only moderate specificity. Laboratory methods for the diagnosis of A. fumigatus include mainly direct microscopic examination, histopathological studies, and serum galactomannan (GM) and (1¡3)-␤-D-glucan assays. The measurement of galactomannan in bronchoalveolar lavage (BAL) fluid shows promise for enhancing the diagnosis of A. fumigatus (4-7), but only approximately 50% of BAL fluid measurement results can match the results of culture and direct examination (8).PCR-based molecular detection systems for fast and sensitive identification by examination of small amounts of DNA in serum and blood have been developed (9-11). However, the lack of standardization for critical activities in the methods and the evaluation of clinical samples has hampered the development of PCR-based diagnostic tests for clinical use....

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

Cited by 17 publications

References 26 publications

Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

Plesiomonas shigelloides Revisited

Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus

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