Rapid Diagnosis of Human Herpesvirus 6 Infection by a Novel DNA Amplification Method, Loop-Mediated Isothermal Amplification

Ihira, Masaru; Yoshikawa, Tetsushi; Enomoto, Yoshihiko; Akimoto, Shiho; Ohashi, Masahiro; Suga, S.; Nishimura, Naoko; Ozaki, Takao; Nishiyama, Yukihiro; Notomi, Tsugunori; Ohta, Yoshinori; Asano, Yoshizo

doi:10.1128/jcm.42.1.140-145.2004

Cited by 94 publications

(60 citation statements)

References 31 publications

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“…LAMP has been used to detect many kinds of virus infection, mainly for DNA virus or bacterial genome infection (4,6,10,11). Recently, RT-LAMP was used to detect West Nile virus (17).…”

Section: Discussionmentioning

confidence: 99%

Rapid Diagnostic Method for Detection of Mumps Virus Genome by Loop-Mediated Isothermal Amplification

et al. 2005

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Most mumps patients are clinically diagnosed without any virological examinations, but some diagnosed cases of mumps may be caused by other pathogens or secondary vaccine failure (SVF). To clarify these issues, a sensitive, specific, and rapid diagnostic method is required. We obtained 60 salivary swabs from 34 patients with natural infection during the course of the illness, 10 samples from patients with vaccine-associated parotitis, and 5 samples from patients with SVF. Total RNA was extracted and subjected to reverse transcription-PCR (RT-PCR) and loop-mediated isothermal amplification (LAMP) for genome amplification. We detected mumps virus RNA corresponding to 0.1 PFU by LAMP within 60 min after RNA extraction, with the same sensitivity as RT-nested PCR. Mumps virus was isolated in 30 of 33 samples within day 2, and mumps virus genome was amplified by LAMP in 32 of them. The quantity of virus titer was calculated by monitoring the time to reach the threshold of turbidity. The viral load decreased after day 3 and was lower in patients serologically diagnosed as having SVF with milder illness. Accuracy of LAMP for the detection of mumps virus genome was confirmed; furthermore, it is of benefit for calculating the viral load, which reflects disease pathogenesis.

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“…LAMP has been used to detect many kinds of virus infection, mainly for DNA virus or bacterial genome infection (4,6,10,11). Recently, RT-LAMP was used to detect West Nile virus (17).…”

Section: Discussionmentioning

confidence: 99%

Rapid Diagnostic Method for Detection of Mumps Virus Genome by Loop-Mediated Isothermal Amplification

et al. 2005

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“…There is a need for the development of sensitive, simple, costeffective, and rapid diagnostic techniques for HCV detection. In 2000, Notomi et al [17] reported a novel nucleic acid amplification method, termed the loop-mediated isothermal amplification (LAMP), that amplifies target sequences with high specificity, efficiency, and speed under isothermal conditions [5,10]. The only equipment needed for the LAMP reaction is a regular laboratory water bath or a heat block that furnishes a constant temperature of 62-65°C [16,17].…”

Section: Introductionmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Hepatitis C virus

et al. 2012

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“…This method also exhibits extremely high amplification efficiency, due in part to its isothermal nature; as there is no time lost due to changes in temperature and the reaction can be conducted at the optimal temperature for enzyme function, the inhibition reactions that often occur at later stages of typical PCR amplifications are less likely to occur. Thus, this method could potentially be a valuable tool for the rapid diagnosis of infectious diseases (5,8,9,12,19,23) in both commercial and hospital laboratories. In this study, we sought to establish a LAMP-based HSV typespecific DNA amplification method and examine its reliability for the detection of HSV DNA from clinical specimens.…”

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confidence: 99%

Rapid Diagnosis of Herpes Simplex Virus Infection by a Loop-Mediated Isothermal Amplification Method

et al. 2005

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Primers for herpes simplex virus type 1 (HSV 1)-specific loop-mediated isothermal amplification (LAMP) method amplified HSV-1 DNA, while HSV-2-specific primers amplified only HSV-2 DNA; no LAMP products were produced by reactions performed with other viral DNAs. The sensitivities of the HSV-1-and HSV-2-specific LAMP methods, determined by agarose gel electrophoresis, reached 500 and 1,000 copies/tube, respectively. The turbidity assay, however, determined the sensitivity of the HSV-1-and HSV-2-specific LAMP methods to be 1,000 and 10,000 copies/tube, respectively. After initial validation studies, 18 swab samples (in sterilized water) collected from patients with either gingivostomatitis or vesicular skin eruptions were examined. HSV-1 LAMP products were detected by agarose gel electrophoresis in the 10 samples that also demonstrated viral DNA detection by real-time PCR. Nine of these 10 samples exhibited HSV-1 LAMP products by turbidity assay. Furthermore, both the agarose gel electrophoresis and the turbidity assay directly detected HSV-1 LAMP products in 9 of the 10 swab samples collected in sterilized water. Next, we examined the reliability of HSV type-specific LAMP for the detection of viral DNA in clinical specimens (culture medium) collected from genital lesions. HSV-2 was isolated from all of the samples and visualized by either agarose gel electrophoresis or turbidity assay.Viral isolation and serological assays are standard methods of herpes simplex virus (HSV) diagnosis. Both viral isolation and serological testing, however, require substantial time to obtain accurate final results. More rapid detection has been achieved by modification of cell culture techniques by centrifugation of inocula on cell monolayers and the use of immunofluorescence techniques (6). Recent studies have suggested that detection of HSV DNA by PCR increases the sensitivity of viral infection detection compared to antigenic detection or cell culture methods (3,4,11,13,14). While quantitative analysis of viral DNA by real-time PCR may become a valuable tool for bedside monitoring of HSV infection and progression (1, 2, 7, 10, 17, 21, 22), it has not yet become a common procedure in hospital laboratories due to the requirement of specific expensive equipment (a thermal cycler).Recently, Notomi et al. (18) reported a novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which is used to amplify DNA under isothermal conditions with high specificity, efficiency, and speed. The most significant advantage of LAMP is the ability to amplify specific sequences of DNA between 63 and 65°C without thermocycling. Thus, the technique requires only simple and cost-effective equipment amenable to use in hospital laboratories. The LAMP method also exhibits both high specificity and high amplification efficiency. As the LAMP method uses four primers which recognize six distinct target DNA sequences, the specificity is extremely high. This method also exhibits extremely high amplification efficiency, due in...

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Rapid Diagnosis of Human Herpesvirus 6 Infection by a Novel DNA Amplification Method, Loop-Mediated Isothermal Amplification

Cited by 94 publications

References 31 publications

Rapid Diagnostic Method for Detection of Mumps Virus Genome by Loop-Mediated Isothermal Amplification

Rapid Diagnostic Method for Detection of Mumps Virus Genome by Loop-Mediated Isothermal Amplification

Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Hepatitis C virus

Rapid Diagnosis of Herpes Simplex Virus Infection by a Loop-Mediated Isothermal Amplification Method

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