Discriminating between varicella-zoster virus vaccine and wild-type strains by loop-mediated isothermal amplification.
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.
Higashimoto
Yuki
Y
Department of Clinical Immunology, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan.
Ihira
Masaru
M
Ohta
Akane
A
Inoue
Shigeki
S
Usui
Chie
C
Asano
Yoshizo
Y
Yoshikawa
Tetsushi
T
D001483:Base Sequence; D002644:Chickenpox / Q000821:virology*; D019433:Chickenpox Vaccine; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D014645:Herpesvirus 3, Human / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009391:Nephelometry and Turbidimetry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D015534:Trans-Activators / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics
D019433:Chickenpox Vaccine; D017931:DNA Primers; D004279:DNA, Viral; C074221:IE62 protein, Human herpesvirus 3; D017874:Immediate-Early Proteins; D015534:Trans-Activators; D014759:Viral Envelope Proteins; C025651:CCCGGG-specific type II deoxyribonucleases; C035337:CCGCGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific