Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Trypanosoma brucei rhodesiense
被引:222
作者:
Njiru, Zablon Kithinji
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Murdoch Univ, Sch Nursing, Mandurah, WA, Australia
Kenya Agr Res Inst, Trypanosomiasis Res Ctr, Kikuyu, KenyaMurdoch Univ, Sch Nursing, Mandurah, WA, Australia
Njiru, Zablon Kithinji
[1
,2
]
Mikosza, Andrew Stanislaw John
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Murdoch Univ, Sch Vet & Biomed Sci, WHO Collaborating Ctr Mol Epidemiol Parasit Infec, Murdoch, WA 6150, AustraliaMurdoch Univ, Sch Nursing, Mandurah, WA, Australia
Mikosza, Andrew Stanislaw John
[3
]
Armstrong, Tanya
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机构:
Murdoch Univ, Sch Vet & Biomed Sci, WHO Collaborating Ctr Mol Epidemiol Parasit Infec, Murdoch, WA 6150, AustraliaMurdoch Univ, Sch Nursing, Mandurah, WA, Australia
Armstrong, Tanya
[3
]
Enyaru, John Charles
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Makerere Univ, Fac Sci, Dept Biochem, Kampala, UgandaMurdoch Univ, Sch Nursing, Mandurah, WA, Australia
Enyaru, John Charles
[4
]
Ndung'u, Joseph Mathu
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FIND, Cointrin, SwitzerlandMurdoch Univ, Sch Nursing, Mandurah, WA, Australia
Ndung'u, Joseph Mathu
[5
]
Thompson, Andrew Richard Christopher
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Murdoch Univ, Sch Vet & Biomed Sci, WHO Collaborating Ctr Mol Epidemiol Parasit Infec, Murdoch, WA 6150, AustraliaMurdoch Univ, Sch Nursing, Mandurah, WA, Australia
Thompson, Andrew Richard Christopher
[3
]
机构:
[1] Murdoch Univ, Sch Nursing, Mandurah, WA, Australia
[2] Kenya Agr Res Inst, Trypanosomiasis Res Ctr, Kikuyu, Kenya
[3] Murdoch Univ, Sch Vet & Biomed Sci, WHO Collaborating Ctr Mol Epidemiol Parasit Infec, Murdoch, WA 6150, Australia
[4] Makerere Univ, Fac Sci, Dept Biochem, Kampala, Uganda
Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62 degrees C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was similar to 1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions.
机构:
Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, JapanKitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Ikadai, H
Tanaka, H
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Tanaka, H
Shibahara, N
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Shibahara, N
Matsuu, A
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Matsuu, A
Uechi, M
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Uechi, M
Itoh, N
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Itoh, N
Oshiro, S
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Oshiro, S
Kudo, N
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Kudo, N
Igarashi, I
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Igarashi, I
Oyamada, T
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
机构:
Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, JapanKitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Ikadai, H
Tanaka, H
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h-index: 0
机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Tanaka, H
Shibahara, N
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Shibahara, N
Matsuu, A
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Matsuu, A
Uechi, M
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Uechi, M
Itoh, N
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h-index: 0
机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Itoh, N
Oshiro, S
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h-index: 0
机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Oshiro, S
Kudo, N
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Kudo, N
Igarashi, I
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan
Igarashi, I
Oyamada, T
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机构:Kitasato Univ, Sch Vet Med & Anim Sci, Dept Vet Parasitol, Towada, Aomori 0348628, Japan