Detection of Human Herpesvirus 7 DNA by Loop-Mediated Isothermal Amplification

Yoshikawa, Tetsushi; Ihira, Masaru; Akimoto, Shiho; Usui, Chie; Miyake, Fumi; Suga, S.; Enomoto, Yoshihiko; Suzuki, Ryota; Nishiyama, Yukihiro; Asano, Yoshizo

doi:10.1128/jcm.42.3.1348-1352.2004

Cited by 59 publications

(33 citation statements)

References 21 publications

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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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“…Although the capability to distinguish between HSV-1 and HSV-2 infection is not crucial for correct administration of antiviral drugs, this discrimination is important from an epidemiological or public health standpoint. As a consequence of the experiment used for determination of the assay sensitivity, it was suggested that the turbidity assay is a less sensitive detection method than agarose gel electrophoresis, as previously suggested (23). However, the turbidity assay is more appropriate for bedside monitoring due to its ease and rapidity.…”

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confidence: 99%

“…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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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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“…The technique, termed loop-mediated isothermal amplification (LAMP), requires a set of conditions and primers different from those used for PCRs (8,12,14,15). LAMP assays have now been reported for several pathogens (8,9,12,14,15,18,20). The reaction typically occurs over 30 to 60 min under isothermal conditions with temperatures ranging from 60 to 65°C and can be conducted with a simple heating block.…”

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Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of BK Virus

et al. 2007

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Loop-mediated isothermal amplification (LAMP) is a novel method for rapid amplification of DNA. Its advantages include rapidity and minimal equipment requirement. The LAMP assay was developed for BK virus (BKV), which is a leading cause of morbidity in renal transplant recipients. The characteristics of the assay, including its specificity and sensitivity, were evaluated. BKV LAMP was performed using various incubation times with a variety of specimens, including unprocessed urine and plasma samples. A ladder pattern on gel electrophoresis, typical of successful LAMP reactions, was observed specifically only for BKV and not for other viruses. The sensitivity of the assay with 1 h of incubation was 100 copies/tube of a cloned BKV fragment. Additionally, a positive reaction was visually ascertained by a simple color reaction using SYBR green dye. BKV LAMP was also successful for urine and plasma specimens without the need for DNA extraction. Due to its simplicity and specificity, the LAMP assay can potentially be developed for "point of care" screening of BKV.

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Detection of Human Herpesvirus 7 DNA by Loop-Mediated Isothermal Amplification

Cited by 59 publications

References 21 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 Diagnosis of Herpes Simplex Virus Infection by a Loop-Mediated Isothermal Amplification Method

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of BK Virus

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