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

Abstract

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...

Rapid Visual Detection of Highly Pathogenic Streptococcus suis Serotype 2 Isolates by Use of Loop-Mediated Isothermal Amplification

“…2A), which is lower than that of gel electrophoresis. This result was consistent with that of another published study (27). However, in the SalK-LAMP reaction, the turbidity detection showed a sensitivity of 7.16 copies/reaction (Fig.…”

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

“…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].…”

Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Diagnosis of Bordetella pertussis Infection

“…The significant advantages of the LAMP method are (i) high amplification efficiency under isothermal conditions (63 to 65°C) and (ii) visual judgment based on the turbidity or fluorescence of the reaction mixture, which is kept in the reaction tube (18). Although it has thus emerged as a powerful tool to facilitate genetic testing for the rapid diagnosis of several viral and bacterial infectious diseases in clinical laboratories (5,11,13,14), the LAMP method has not been evaluated for the diagnosis of B. pertussis infection. In the present study, we developed a LAMP method for diagnosis of B. pertussis infection and evaluated its sensitivity, specificity, and applicability for clinical specimens.…”