Rapid Detection of the Severe Acute Respiratory Syndrome (SARS) Coronavirus by a Loop-Mediated Isothermal Amplification Assay

Poon, Leo L. M.; Leung, Cynthia Sau-Wai; Tashiro, Masato; Chan, Kwok Hung; Wong, Bonnie Wing Yan; Yuen, Kwok‐Yung; Guan, Yi; Peiris, J. S. Malik

doi:10.1373/clinchem.2004.032011

Cited by 113 publications

(104 citation statements)

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“…An accurate and rapid diagnosis system for periodontitis is essential for periodontal treatment. Several recent reports have demonstrated the usefulness of the LAMP method (10,11,17,26,27,33,49). Therefore, we focused on the LAMP method for the rapid detection of periodontopathic bacteria.…”

Section: Discussionmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification Method for Rapid Detection of the Periodontopathic BacteriaPorphyromonas gingivalis,Tannerella forsythia, andTreponema denticola

et al. 2005

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Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the rapid detection of the major periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. The LAMP method amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. In this study, we initially designed the primers for LAMP assays to detect these bacteria and evaluated the specificity and sensitivity of these assays. The specificities of the primers for these bacteria were examined using various oral bacteria and various reaction times. The lower detection limits of the 60-min LAMP reaction without loop primers were 1 g/tube for P. gingivalis, 10 fg/tube for T. forsythia, and 1 ng/tube for T. denticola. Addition of the loop primers for each bacterium improved the detection specificities and sensitivities by several magnitudes. Furthermore, LAMP assays were applied to the rapid detection of these periodontal pathogens in clinical specimens, and the results were compared with those of conventional PCR detection. The results of the LAMP assays corresponded to those of conventional PCR assays. These results indicate that the LAMP assay is an extremely rapid, highly sensitive, specific method. This method is very useful for the rapid detection of periodontopathic bacteria and the diagnosis of periodontal disease.

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Section: Discussionmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification Method for Rapid Detection of the Periodontopathic BacteriaPorphyromonas gingivalis,Tannerella forsythia, andTreponema denticola

et al. 2005

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“…In contrast to Taq DNA polymerase used in PCR, the Bst DNA polymerase is resistant to inhibitors present in crude biological samples (Kaneko et al, 2007). As four or six specific primers that recognize six or eight distinct sequences on the target DNA are required, LAMP has been shown to amplify target DNA with high specificity and is widely used in the clinical diagnosis of epidemic bacteria Knight et al, 2014), viruses (Poon et al, 2004;Curtis et al, 2014), parasites (Li et al, 2012;Ni et al, 2014) and fetal sex identification (Fu et al, 2011), as well as detection of the hypervirulent strain and toxin types of C. difficile (Kato et al, 2005;Norén et al, 2011;Boyanton et al, 2012). In the current study, five sets of LAMP primers were designed and the assay was optimized for detection of ermB.…”

Section: Introductionmentioning

confidence: 99%

Rapid detection of ermB gene in Clostridium difficile by loop-mediated isothermal amplification

Lin

Liu

Wang

et al. 2015

Journal of Medical Microbiology

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Macrolide-lincosamide-streptogramin B resistance in Clostridium difficile is mostly due to the ermB resistance determinant. Here, we describe a sensitive and rapid molecular method to detect ermB in C. difficile to contribute to the wider epidemiological study. Five sets of loop-mediated isothermal amplification (LAMP) primers were designed and optimized for rapid detection of ermB. The specificity and sensitivity of the primers for ermB were detected, and the ermB LAMP assay was compared to conventional PCR with 80 clinical isolates of C. difficile. Real-time monitoring of turbidity and chromogenic reaction were used to determine negative and positive results. A total of 26 pathogenic bacterial strains of different species were found to be negative for ermB, which indicated the high specificity of the primers. ermB was detected in 78.8 % (63/80) of the clinical isolates by both LAMP and conventional PCR. The detection limit of LAMP was 36.1 pg DNA ml 21 and its sensitivity was 10-fold greater than that of conventional PCR. This study is the first report regarding the development and application of the LAMP assay for detection of the ermB gene in C. difficile strains. The developed LAMP method is sensitive, specific and provides a user-friendly visual approach for the rapid detection of ermB-bearing C. difficile. Received 27 March 2015Accepted 13 June 2015 INTRODUCTIONClostridium difficile is a causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis, usually as a consequence of antimicrobial therapy. During the last few decades, C. difficile infection (CDI) has become more severe, more recurrent, more resistant to antibiotics and more refractory to standard treatment, which poses a significant burden on patients, healthcare systems and society in general. Estimates suggest that the annual costs for management of CDI amount to more than $3 billion in the USA and J3 billion in Europe (Bouza, 2012;McGlone et al., 2012). Major risk factors for CDI include hospitalization, advanced age and antibiotic exposure, the latter including clindamycin and erythromycin (Aziz et al., 2001;Gerding, 1989;Kelly et al., 1994;Slimings & Riley, 2014). The majority of epidemic isolates are resistant to clindamycin, erythromycin, broad-spectrum cephalosporins and fluoroquinolones (Johnson et al., 1999; Spigaglia et al., 2011;Slimings & Riley, 2014). Thus, resistance to clindamycin and erythromycin, two antibiotics of the macrolidelincosamide-streptogramin B (MLSB) group, would further increase the risk of CDI.In C. difficile, resistance to the MLSB group of antibiotics is mainly encoded by the ermB resistance determinant. The ermB gene encodes a 23S rRNA methyltransferase that modifies the target site for the antibiotic and is located on a specific mobilizable non-conjugative element, Tn5398 (Farrow et al., 2001). However, the ermB gene is located on elements other than Tn5398 in the majority of clinical isolates. In total, 17 genetic organizations of such elements 3These authors contributed equally to this work...

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“…Nevertheless, it may generate a false positive product easily, which results from amplicon handling and the primer dimers; additionally, the use of primer at a high concentration can also inhibit LAMP amplification (Njiru et al 2008). Several previous researchers have made efforts to develop RT-LAMP assay, such as using the restriction enzyme to digest RT-LAMP products (Curtis et al 2008), establishing one step reaction to decline the risk of cross-contamination to the minimum (Poon et al 2004). By contrast, the fact that RT-PCR needs a well-equipped laboratory and multiple reaction steps makes cross-contamination less possible to take place.…”

Section: Discussionmentioning

confidence: 99%

Comparison of reverse-transcription loop-mediated isothermal amplification and reverse-transcription polymerase chain reaction for grass carp reovirus

2015

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Grass carp reovirus (GCRV) has been assigned to a newly established Aquareovirus genus in the family of Reoviridae which leads to haemorrhagic disease and extremely high mortality rate in grass carp. In this study, comparison was made between the novel one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) and the reverse transcription polymerase chain reaction (RT-PCR) for detection of grass carp reovirus. The result indicated that RT-LAMP had × 10 higher sensitivity comparable to RT-PCR. The specificity of the two methods for GCRV detection were both developed successfully by other three aquatic viruses. In the field trial, both RT-PCR and RT-LAMP methods were applied to detect the samples from different infected organs and tissues. The result demonstrated that RT-LAMP had a high accuracy to confirm the diagnosis as well as the RT-PCR. This study showed that the RT-LAMP, compared to the RT-PCR, was simple, time-saving, convenient, but required specificity primers and possibly generated false positive product. Its products, unlike RT-PCR, could not be direcly used in further molecular research after purification. Thus RT-LAMP might be an optimal diagnostic method for rapid and preliminary diagnosis of GCRV infection in resource-limited setting situation.

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Rapid Detection of the Severe Acute Respiratory Syndrome (SARS) Coronavirus by a Loop-Mediated Isothermal Amplification Assay

Cited by 113 publications

References 19 publications

Loop-Mediated Isothermal Amplification Method for Rapid Detection of the Periodontopathic BacteriaPorphyromonas gingivalis,Tannerella forsythia, andTreponema denticola

Loop-Mediated Isothermal Amplification Method for Rapid Detection of the Periodontopathic BacteriaPorphyromonas gingivalis,Tannerella forsythia, andTreponema denticola

Rapid detection of ermB gene in Clostridium difficile by loop-mediated isothermal amplification

Comparison of reverse-transcription loop-mediated isothermal amplification and reverse-transcription polymerase chain reaction for grass carp reovirus

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