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Journal of Clinical Microbiology, August 2008, p. 2665-2670, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00216-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Discriminating between Varicella-Zoster Virus Vaccine and Wild-Type Strains by Loop-Mediated Isothermal Amplification{triangledown}

Yuki Higashimoto,1,4 Masaru Ihira,2 Akane Ohta,3 Shigeki Inoue,1 Chie Usui,4 Yoshizo Asano,3,4 and Tetsushi Yoshikawa3,4*

Department of Clinical Immunology, Fujita Health University School of Health Sciences,1 Department of Medical Information Technology, Fujita Health University College,2 21st Century COE Research Center, Fujita Health University,3 Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi, Japan4

Received 3 February 2008/ Returned for modification 17 May 2008/ Accepted 2 June 2008

The loop-mediated isothermal amplification (LAMP) method was developed to distinguish between the varicella-zoster virus (VZV) vaccine (vOka) strain and wild-type strains. Two single nucleotide polymorphisms (SNPs) (nucleotide [nt] 105705 for VR-1 VZV LAMP and nt 106262 for VR-2 VZV LAMP) located in the open reading frame 62 gene were selected as LAMP targets. Amplified vOka DNA demonstrated a typical ladder pattern; however, no LAMP product was detected in reactions performed with DNAs from other human herpesviruses by either VR-1 VZV LAMP or VR-2 VZV LAMP. This result was confirmed by a turbidity assay. The sensitivities of both VR-1 and VR-2 VZV LAMP determined by either the turbidity assay or agarose gel electrophoresis were 100 copies per reaction. To discriminate the vOka strain from wild-type strains, VR-1 and VR-2 VZV LAMP products were digested with the appropriate restriction enzymes (SacII for VR-1 LAMP and SmaI for VR-2 LAMP). The digested products were clearly different in the vOka strain and wild-type strains. To evaluate the utility of the LAMP methods for rapid differentiation, viral DNA (without DNA extraction) in swab samples was directly tested. Wild-type VZV DNA was detected in 20 swab samples by either VR-1 VZV LAMP or VR-2 VZV LAMP. Sequence analysis confirmed the expected SNPs in the LAMP products amplified from the vOka strain and the five wild-type strains.

* Corresponding author. Mailing address: Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi 4701192, Japan. Phone: 81-562-939251. Fax: 81-562-952216. E-mail: tetsushi{at}fujita-hu.ac.jp

{triangledown} Published ahead of print on 11 June 2008.

Journal of Clinical Microbiology, August 2008, p. 2665-2670, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00216-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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