論文 - 太田 茜
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Cold Tolerance in the Nematode Caenorhabditis elegans 招待あり 査読あり
Kuhara A., Takagaki N., Okahata M., Ohta A.
Adv Exp Med Biol. 1461 33 - 46 2024年7月
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Ohta A., Sato Y., Isono K., Kajino T., Tanaka K., Taji T., Kuhara A.
PNAS nexus 3 ( 8(293) ) 1 - 13 2024年7月
担当区分：筆頭著者, 責任著者
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Ohnishi K, Sokabe T, Miura T, Tominaga M, Ohta A, Kuhara A
Nature commun. 15 ( 1660 ) 1 - 13 2024年2月
担当区分：責任著者
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Okahata M, Sawada N, Nakao K, Ohta A, Kuhara A
Scientific Reports 14 ( 5401 ) 1 - 14 2024年3月
担当区分：責任著者
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Haruka Motomura, Makoto Ioroi, Kazutoshi Murakami, Atsushi Kuhara, Akane Ohta
PNAS 119 ( 32 ) 1 - 9 2022年8月
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Ohnishi K, Saito S, Miura Y, Ohta A, Tominaga M, Sokabe T, Kuhara A
Scientific Reports 10 ( 18566 ) 1 - 14 2020年10月
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The mechanoreceptor DEG-1 regulates cold tolerance in Caenorhabditis elegans. 査読あり 国際誌
Natsune Takagaki, Akane Ohta, Kohei Ohnishi, Akira Kawanabe, Yohei Minakuchi, Atsushi Toyoda, Yuichiro Fujiwara, Atsushi Kuhara
EMBO reports e48671 ( Article ) e48671 - 12 2020年2月
担当区分：責任著者
Caenorhabditis elegans mechanoreceptors located in ASG sensory neurons have been found to sense ambient temperature, which is a key trait for animal survival. Here, we show that experimental loss of xanthine dehydrogenase (XDH-1) function in AIN and AVJ interneurons results in reduced cold tolerance and atypical neuronal response to changes in temperature. These interneurons connect with upstream neurons such as the mechanoreceptor-expressing ASG. Ca2+ imaging revealed that ASG neurons respond to warm temperature via the mechanoreceptor DEG-1, a degenerin/epithelial Na+ channel (DEG/ENaC), which in turn affects downstream AIN and AVJ circuits. Ectopic expression of DEG-1 in the ASE gustatory neuron results in the acquisition of warm sensitivity, while electrophysiological analysis revealed that DEG-1 and human MDEG1 were involved in warm sensation. Taken together, these results suggest that cold tolerance is regulated by mechanoreceptor-mediated circuit calculation.
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Cold acclimation via the KQT-2 potassium channel is modulated by oxygen in Caenorhabditis elegans. 査読あり 国際共著 国際誌
Misaki Okahata, Aguan D Wei, Akane Ohta, Atsushi Kuhara
Science advances 5 ( 2 ) eaav3631 - 12 2019年2月
担当区分：責任著者
Adaptive responses to external temperatures are essential for survival in changing environments. We show here that environmental oxygen concentration affects cold acclimation in Caenorhabditis elegans and that this response is regulated by a KCNQ-type potassium channel, KQT-2. Depending on culture conditions, kqt-2 mutants showed supranormal cold acclimation, caused by abnormal thermosensation in ADL chemosensory neurons. ADL neurons are responsive to temperature via transient receptor potential channels-OSM-9, OCR-2, and OCR-1-with OCR-1 negatively regulating ADL function. Similarly, KQT-2 and KQT-3 regulate ADL activity, with KQT-2 positively regulating ADL function. Abnormal cold acclimation and acute temperature responses of ADL neurons in kqt-2 mutants were suppressed by an oxygen-receptor mutation in URX coelomic sensory neurons, which are electrically connected to ADL via RMG interneurons. Likewise, low oxygen suppressed supranormal kqt-2 cold acclimation. These data thus demonstrate a simple neuronal circuit integrating two different sensory modalities, temperature and oxygen, that determines cold acclimation.
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Ujisawa T, Ohta A, Ii T, Minakuchi Y, Toyoda A, Ii M, Kuhara A
PNAS 115 ( 35 ) 8823 - 8828 2018年
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Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis elegans 査読あり
Tomoyo Ujisawa, Akane Ohta, Misato Uda-Yagi, Atsushi Kuhara
PLOS ONE 11 ( 10 ) 1 - 20 2016年10月
担当区分：責任著者 出版者・発行元：PUBLIC LIBRARY SCIENCE
Temperature sensation by the nervous system is essential for life and proliferation of animals. The molecular-physiological mechanisms underlying temperature signaling have not been fully elucidated. We show here that diverse regulatory machinery underlies temperature sensation through trimeric G-protein signaling in the nematode Caenorhabditis elegans. Molecular-genetic studies demonstrated that cold tolerance is regulated by additive functions of three Ga proteins in a temperature-sensing neuron, ASJ, which is also known to be a light-sensing neuron. Optical recording of calcium concentration in ASJ upon temperature-changes demonstrated that three Ga proteins act in different aspects of temperature signaling. Calcium concentration changes in ASJ upon temperature change were unexpectedly decreased in a mutant defective in phosphodiesterase, which is well known as a negative regulator of calcium increase. Together, these data demonstrate commonalities and differences in the molecular components concerned with light and temperature signaling in a single sensory neuron.
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Sperm Affects Head Sensory Neuron in Temperature Tolerance of Caenorhabditis elegans 査読あり
Satoru Sonoda, Akane Ohta, Ayana Maruo, Tomoyo Ujisawa, Atsushi Kuhara
Cell reports 16 ( 1 ) 56 - 65 2016年6月
担当区分：責任著者 出版者・発行元：CELL PRESS
Tolerance to environmental temperature change is essential for the survival and proliferation of animals. The process is controlled by various body tissues, but the orchestration of activity within the tissue network has not been elucidated in detail. Here, we show that sperm affects the activity of temperature-sensing neurons (ASJ) that control cold tolerance in Caenorhabditis elegans. Genetic impairment of sperm caused abnormal cold tolerance, which was unexpectedly restored by impairment of temperature signaling in ASJ neurons. Calcium imaging revealed that ASJ neuronal activity in response to temperature was decreased in sperm mutant gsp-4 with impaired protein phosphatase 1 and rescued by expressing gsp-4 in sperm. Genetic analysis revealed a feedback network in which ASJ neuronal activity regulates the intestine through insulin and a steroid hormone, which then affects sperm and, in turn, controls ASJ neuronal activity. Thus, we propose that feedback between sperm and a sensory neuron mediating temperature tolerance.
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Light and pheromone-sensing neurons regulates cold habituation through insulin signalling in Caenorhabditis elegans 査読あり
Akane Ohta, Tomoyo Ujisawa, Satoru Sonoda, Atsushi Kuhara
Nature communications 5 ( 4412 ) 1 - 12 2014年7月
担当区分：筆頭著者, 責任著者 出版者・発行元：NATURE PUBLISHING GROUP
Temperature is a critical environmental stimulus that has a strong impact on an organism's biochemistry. Animals can respond to changes in ambient temperature through behaviour or altered physiology. However, how animals habituate to temperature is poorly understood. The nematode C. elegans stores temperature experiences and can induce temperature habituation-linked cold tolerance. Here we show that light and pheromone-sensing neurons (ASJ) regulate cold habituation through insulin signalling. Calcium imaging reveals that ASJ neurons respond to temperature. Cold habituation is abnormal in a mutant with impaired cGMP signalling in ASJ neurons. Insulin released from ASJ neurons is received by the intestine and neurons regulating gene expression for cold habituation. Thus, temperature sensation in a light and pheromone-sensing neuron produces a robust effect on insulin signalling that controls experience-dependent temperature habituation.
DOI： 10.1038/ncomms5412
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Herpes simplex virus type 1 UL14 tegument protein regulates intracellular compartmentalization of major tegument protein VP16 査読あり
Akane Ohta, Yohei Yamauchi, Yoshifumi Muto, Hiroshi Kimura, Yukihiro Nishiyama
Virology Journal 8 ( 365 ) 2011年7月
担当区分：筆頭著者 出版者・発行元：BIOMED CENTRAL LTD
Background: Herpes simplex virus type 1 (HSV-1) has a complicated life-cycle, and its genome encodes many components that can modify the cellular environment to facilitate efficient viral replication. The protein UL14 is likely involved in viral maturation and egress (Cunningham C. et al), and it facilitates the nuclear translocation of viral capsids and the tegument protein VP16 during the immediate-early phase of infection (Yamauchi Y. et al, 2008). UL14 of herpes simplex virus type 2 exhibits multiple functions (Yamauchi Y. et al, 2001, 2002, 2003).
Methods: To better understand the function(s) of UL14, we generated VP16-GFP-incorporated UL14-mutant viruses with either single (K51M) or triple (R60A, R64A, E68D) amino acid substitutions in the heat shock protein (HSP)-like sequence of UL14. We observed the morphology of cells infected with UL14-null virus and amino acid-substituted UL14-mutant viruses at different time points after infection.
Results: UL14(3P)-VP16GFP and UL14D-VP16GFP (UL14-null) viruses caused similar defects with respect to growth kinetics, compartmentalization of tegument proteins, and cellular morphology in the late phase. Both the UL14D-VP16GFP and UL14(3P)-VP16GFP viruses led to the formation of an aggresome that incorporated some tegument proteins but did not include nuclear-egressed viral capsids.
Conclusions: Our findings suggest that a cluster of charged residues within the HSP-like sequence of UL14 is important for the molecular chaperone-like functions of UL14, and this activity is required for the acquisition of functionality of VP16 and UL46. In addition, UL14 likely contributes to maintaining cellular homeostasis following infection, including cytoskeletal organization. However, direct interactions between UL14 and VP16, UL46, or other cellular or viral proteins remain unclear. -
Novel and Conserved Protein Macoilin Is Required for Diverse Neuronal Functions in Caenorhabditis elegans 査読あり
Akiko Miyara, Akane Ohta, Yoshifumi Okochi, Yuki Tsukada, Atsushi Kuhara, Ikue Mori
PLOS Genetics 7 ( 5 ) 2011年5月
出版者・発行元：PUBLIC LIBRARY SCIENCE
Neural signals are processed in nervous systems of animals responding to variable environmental stimuli. This study shows that a novel and highly conserved protein, macoilin (MACO-1), plays an essential role in diverse neural functions in Caenorhabditis elegans. maco-1 mutants showed abnormal behaviors, including defective locomotion, thermotaxis, and chemotaxis. Expression of human macoilin in the C. elegans nervous system weakly rescued the abnormal thermotactic phenotype of the maco-1 mutants, suggesting that macoilin is functionally conserved across species. Abnormal thermotaxis may have been caused by impaired locomotion of maco-1 mutants. However, calcium imaging of AFD thermosensory neurons and AIY postsynaptic interneurons of maco-1 mutants suggest that macoilin is required for appropriate responses of AFD and AIY neurons to thermal stimuli. Studies on localization of MACO-1 showed that C. elegans and human macoilins are localized mainly to the rough endoplasmic reticulum. Our results suggest that macoilin is required for various neural events, such as the regulation of neuronal activity.
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Mitochondria and viruses 招待あり 査読あり
Akane Ohta, Yukihiro Nishiyama
Mitochondrion 11 ( 1 ) 1 - 12 2011年1月
担当区分：筆頭著者 出版者・発行元：ELSEVIER SCI LTD
Mitochondria are involved in a variety of cellular metabolic processes, and their functions are regulated by extrinsic and intrinsic stimuli including viruses. Recent studies have shown that mitochondria play a central role in the primary host defense mechanisms against viral infections, and a number of novel viral and mitochondrial proteins are involved in these processes. Some viral proteins localize in mitochondria and interact with mitochondrial proteins to regulate cellular responses. This review summarizes recent findings on the functions and roles of these molecules as well as mitochondrial responses to viral infections. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
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Recombinant human monoclonal antibodies to human cytomegalovirus glycoprotein B neutralize virus in a complement-dependent manner 査読あり
Akane Ohta, Ayano Fujita, Tsugiya Murayama, Yoshitaka Iba, Yoshikazu Kurosawa, Tetsushi Yoshikawa, Yoshizo Asano
Microbes and Infection 11 ( 13 ) 1029 - 1036 2009年11月
担当区分：筆頭著者 出版者・発行元：ELSEVIER SCIENCE BV
Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used it combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV In a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V(H) region Seven representative clones front these 14 groups had a strong gB654 binding affinity by Surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity In vitro. In contrast, complete human IgG(1) antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate tumoral immunity to the selected pathogen. (C) 2009 Elsevier Masson SAS. All rights reserved.
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Diverse regulation of sensory signaling by C-elegans nPKC-epsilon/eta TTX-4 査読あり
Y Okochi, KD Kimura, A Ohta, Mori, I
EMBO Journal 24 ( 12 ) 2127 - 2137 2005年6月
出版者・発行元：NATURE PUBLISHING GROUP
Molecular and pharmacological studies in vitro suggest that protein kinase C (PKC) family members play important roles in intracellular signal transduction. Nevertheless, the in vivo roles of PKC are poorly understood. We show here that nPKC-epsilon/eta TTX-4 in the nematode Caenorhabditis elegans is required for the regulation of signal transduction in various sensory neurons for temperature, odor, taste, and high osmolality. Interestingly, the requirement for TTX-4 differs in different sensory neurons. In AFD thermosensory neurons, gain or loss of TTX-4 function inactivates or hyperactivates the neural activity, respectively, suggesting negative regulation of temperature sensation by TTX-4. In contrast, TTX-4 positively regulates the signal sensation of ASH nociceptive neurons. Moreover, in AWA and AWC olfactory neurons, TTX-4 plays a partially redundant role with another nPKC, TPA-1, to regulate olfactory signaling. These results suggest that C. elegans nPKCs regulate different sensory signaling in various sensory neurons. Thus, C. elegans provides an ideal model to reveal genetically novel components of nPKC-mediated molecular pathways in sensory signaling.
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Ohta A., Morimoto C., Kamino S., Tezuka M.
Zoological Science 42 ( 1 ) 4 - 12 2024年11月
担当区分：筆頭著者, 責任著者
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Mori Y., Ohta A., Kuhara A.
Neuroscience Research in press 2024年11月
担当区分：責任著者
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Misaki Okahata, Natsumi Sawada, Kenji Nakao, Akane Ohta, Atsushi Kuhara
Research Square 1 - 27 2022年11月
担当区分：責任著者
DOI： 10.21203/rs.3.rs-2261648/v1
その他リンク： https://www.researchsquare.com/article/rs-2261648/v1.html
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Molecular physiology regulating cold tolerance and acclimation of Caenorhabditis elegans 招待あり 査読あり
Misaki OKAHATA, Haruka MOTOMURA, Akane OHTA, Atsushi KUHARA
PJA SerB 98 ( 3 ) 126 - 139 2022年3月
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daf-16/FOXO isoform b in AIY neurons is involved in low preference for Bifidobacterium infantis in Caenorhabditis elegans 査読あり 国際誌
Sun S, Ohta A, Kuhara A, Nishikawa Y, Kage-Nakadai E
Neuroscience Research 150 8 - 16 2020年1月
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Temperature response in cold tolerance of C. elegans 招待あり 査読あり
Kuhara A, Ohta A
Impact 2018 ( 7 ) 44 - 46 2018年10月
担当区分：責任著者
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Molecular and Cellular Network Systems Underlying Cold Tolerance of Caenorhabditis elegans 招待あり 査読あり
Ohnishi K, Takagaki N, Okahata M, Fujita M, Ohta A, Kuhara A
Cryobiology and Cryotechnology 64 ( 2 ) 53 - 59 2018年
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Natural variations of cold tolerance and temperature acclimation in Caenorhabditis elegans 査読あり
Misaki Okahata, Akane Ohta, Hitomi Mizutani, Yohei Minakuchi, Atsushi Toyoda, Atsushi Kuhara
Journal of Comparative Physiology B 186 ( 8 ) 985 - 998 2016年12月
担当区分：筆頭著者, 責任著者 出版者・発行元：SPRINGER HEIDELBERG
Temperature is critical for the survival and proliferation of animals, which must be adapted to cope with environmental temperature changes. In this study, we demonstrated natural variations in the phenotypes of temperature tolerance and temperature acclimation of the nematode Caenorhabditis elegans, and we decoded whole genome sequence of six natural variations, which enabled us to map responsible gene polymorphisms onto specific chromosomal regions. The C. elegans laboratory strain, N2, survives at 2 A degrees C after cultivation at 15 A degrees C but is unable to survive at 2 A degrees C after cultivation at 20 or 25 A degrees C. This cultivation-temperature-dependent cold tolerance occurs within a few hours after the temperature shift and is termed cold acclimation. We measured the cold tolerance and cold acclimation phenotypes of many natural variants isolated from various areas. CB4854 showed weaker cold tolerance associated with gene polymorphisms on the sex chromosome decoded by whole genome sequencing. Variable cold acclimation phenotypes were exhibited in twelve natural isolates and the large difference was seen between CB4856 and AB1 strains. CB4856, isolated from Hawaii, acclimated slowly to a new temperature, whereas AB1, isolated from Australia, acclimated rapidly. By the whole genome sequencing analysis, two different polymorphisms responsible for the accelerated cold acclimation in AB1 were mapped to specific chromosomal regions.
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Caenorhabditis elegans homologue of Prox1/Prospero is expressed in the glia and is required for sensory behavior and cold tolerance 査読あり
Eriko Kage-Nakadai, Akane Ohta, Tomoyo Ujisawa, Simo Sun, Yoshikazu Nishikawa, Atsushi Kuhara, Shohei Mitani
Genes to Cells 21 ( 9 ) 936 - 948 2016年9月
出版者・発行元：WILEY-BLACKWELL
The Caenorhabditis elegans (C.elegans) amphid sensory organ contains only 4 glia-like cells and 24 sensory neurons, providing a simple model for analyzing glia or neuron-glia interactions. To better characterize glial development and function, we carried out RNA interference screening for transcription factors that regulate the expression of an amphid sheath glial cell marker and identified pros-1, which encodes a homeodomain transcription factor homologous to Drosophila prospero/mammalian Prox1, as a positive regulator. The functional PROS-1::EGFP fusion protein was localized in the nuclei of the glia and the excretory cell but not in the amphid sensory neurons. pros-1 deletion mutants exhibited larval lethality, and rescue experiments showed that pros-1 and human Prox1 transgenes were able to rescue the larval lethal phenotype, suggesting that pros-1 is a functional homologue of mammalian Prox1, at least partially. We further found that the structure and functions of sensory neurons, such as the morphology of sensory endings, sensory behavior and sensory-mediated cold tolerance, appeared to be affected by the pros-1 RNAi. Together, our results show that the C.elegans PROS-1 is a transcriptional regulator in the glia but is involved not only in sensory behavior but also in sensory-mediated physiological tolerance.
DOI： 10.1111/gtc.12394
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Cold tolerance assay for studying cultivation-temperature-dependent cold habituation in C. elegans
Ujisawa T, Ohta, A, Okahata M, Sonoda S, Kuhara, A
Protocol Exchange 2014年
担当区分：筆頭著者, 責任著者
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Long-term calcium imaging of ASJ sensoryneuron controlling cold tolerance in C. elegans
Ujisawa T, Ohta, A, Kuhara, A
Protocol Exchange 2014年
担当区分：責任著者
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Molecular mechanism for trimetric G protein-coupled thermosensation and synaptic regulation in the temperature response circuit of Caenorhabditis elegans 査読あり
Akane Ohta, Atsushi Kuhara
Neuroscience Research 76 ( 3 ) 119 - 124 2013年7月
担当区分：筆頭著者, 責任著者 出版者・発行元：ELSEVIER IRELAND LTD
How the nervous system controls the sensation and memory of information from the environment is an essential question. The nematode Caenorhabditis elegans is a useful model for elucidating neural information processing that mediates sensation and memory. The entire nervous system of C. elegans consists of only 302 neurons, and their wiring diagram has been revealed by electron microscopy analysis. Here, we review the molecular and physiological mechanisms responsible for the neural circuit-mediated temperature-seeking behavior (thermotaxis) in C. elegans. Recent molecular biology studies and optogenetic analyses, such as the optical manipulation of neural activity, and neural imaging have revealed the novel concept of neural calculation. Most significantly, trimetric G proteincoupled thermosensation, single sensory neuron-based memory, and the orchestrated synaptic transmission system have been elucidated. (c) 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
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Development of a Human Herpesvirus 6 Species-Specific Immunoblotting Assay 査読あり
Yuki Higashimoto, Akane Ohta, Yukihiro Nishiyama, Masaru Ihira, Ken Sugata, Yoshizo Asano, Daniel L. Peterson, Dharam V. Ablashi, Paolo Lusso, Tetsushi Yoshikawa
Journal of Clinical Microbiology 50 ( 4 ) 1245 - 1251 2012年4月
出版者・発行元：AMER SOC MICROBIOLOGY
In order to assess the full spectrum of human herpesvirus 6A (HHV-6A)- and HHV-6B-associated diseases, we sought to develop an HHV-6 species-specific serological assay based on immunoblot analysis. The immunodominant proteins encoded by open reading frame U11, p100 for HHV-6A (strain U1102) and 101K for HHV-6B (strain Z29), were selected to generate virus species-specific antigens. Recombinant p100 and 101K were produced in a prokaryotic expression system. The expression of these proteins was confirmed by using anti-His tag and 101K-specific monoclonal antibodies. HHV-6 species-specific antibodies were detected by immunoblotting in patient sera. Eighty-seven serum samples obtained from various subjects were utilized to determine the reliability of the method for clinical use. Ten of twelve exanthem subitum convalescent-phase sera reacted exclusively with 101K, whereas none of twelve acute-phase sera reacted with either protein. Two of three sera collected from HHV-6A-infected patients reacted with p100 and 101K. Although all five acute and convalescent-phase sera obtained from transplant recipients reacted exclusively with 101K, two of six convalescent-phase sera obtained from patients with drug-induced hypersensitivity syndrome reacted with both p100 and 101K. Of 38 sera obtained from healthy adults, 31 were positive for 101K antibody, while 4 reacted with both proteins. However, PCR analysis of peripheral blood mononuclear cells and saliva from these subjects did not detect HHV-6A DNA. In conclusion, this novel serological assay based on immunoblot analysis using recombinant HHV-6A p100 and HHV-6B 101K allowed us to discriminate between HHV-6A- and HHV-6B-specific antibodies.
DOI： 10.1128/JCM.05834-11
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Loop-mediated isothermal amplification for discriminating between human herpesvirus 6 A and B 査読あり
Masaru Ihira, Akane Ohta, Ken Sugata, Sadao Suga, Yoshizo Asano, Tetsushi Yoshiakwa
Journal of Virological Methods 154 ( 1-2 ) 223 - 225 2008年12月
出版者・発行元：ELSEVIER SCIENCE BV
Genotyping of human herpesvirus 6 (HHV-6) is important clinically, particularly for the diagnosis of neurological diseases. The objective of this study was to establish a rapid HHV-6 genotyping method using the loop-mediated isothermal amplification (LAMP) method. An Accl site is located in the target sequence of HHV-6 B, but not in HHV-6 A. LAMP products were digested with the Accl enzyme and then separated by agarose gel electrophoresis to differentiate the digest pattern of the two variants. The fragment patterns were clearly different between HHV-6 A and B. In order to evaluate the reliability of this HHV-6 genotyping method for use in the clinical laboratory, serum samples from 20 patients with either primary HHV-6 infection or viral reactivation were collected and analyzed. HHV-6 DNA was amplified directly from the serum samples and all 20 LAMP products were positive for HHV-6 B. (C) 2008 Elsevier B.V. All rights reserved.
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Discriminating between varicella-zoster virus vaccine and wild-type strains by loop-mediated isothermal amplification 査読あり
Yuki Higashimoto, Masaru Ihira, Akane Ohta, Shigeki Inoue, Chie Usui, Yoshizo Asano, Tetsushi Yoshikawa
Journal of Clinical Microbiology 46 ( 8 ) 2665 - 2670 2008年8月
出版者・発行元：AMER SOC MICROBIOLOGY
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.
DOI： 10.1128/JCM.00216-08