Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Trypanosoma brucei rhodesiense

被引:222
作者
Njiru, Zablon Kithinji [1 ,2 ]
Mikosza, Andrew Stanislaw John [3 ]
Armstrong, Tanya [3 ]
Enyaru, John Charles [4 ]
Ndung'u, Joseph Mathu [5 ]
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
[5] FIND, Cointrin, Switzerland
来源
PLOS NEGLECTED TROPICAL DISEASES | 2008年 / 2卷 / 02期
关键词
D O I
10.1371/journal.pntd.0000147
中图分类号
R51 [传染病];
学科分类号
100401 ;
摘要
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.
引用
收藏
页数:8
相关论文
共 23 条
  • [1] Operational feasibility of using loop-mediated isothermal amplification for diagnosis of pulmonary tuberculosis in microscopy centers of developing countries
    Boehme, Catharina C.
    Nabeta, Pamela
    Henostroza, German
    Raqib, Rubhana
    Rahim, Zeaur
    Gerhardt, Martina
    Sanga, Erica
    Hoelscher, Michael
    Notomi, Tsugunori
    Hase, Tetsu
    Perkins, Mark D.
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 2007, 45 (06) : 1936 - 1940
  • [2] A SIMPLE PROCEDURE FOR OPTIMIZING THE POLYMERASE CHAIN-REACTION (PCR) USING MODIFIED TAGUCHI METHODS
    COBB, BD
    CLARKSON, JM
    [J]. NUCLEIC ACIDS RESEARCH, 1994, 22 (18) : 3801 - 3805
  • [3] THE SERUM RESISTANCE-ASSOCIATED (SRA) GENE OF TRYPANOSOMA-BRUCEI-RHODESIENSE ENCODES A VARIANT SURFACE GLYCOPROTEIN-LIKE PROTEIN
    DEGREEF, C
    HAMERS, R
    [J]. MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 1994, 68 (02) : 277 - 284
  • [4] A GENE EXPRESSED ONLY IN SERUM-RESISTANT VARIANTS OF TRYPANOSOMA-BRUCEI-RHODESIENSE
    DEGREEF, C
    IMBERECHTS, H
    MATTHYSSENS, G
    VANMEIRVENNE, N
    HAMERS, R
    [J]. MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 1989, 36 (02) : 169 - 176
  • [5] The origins of a new Trypanosoma brucei rhodesiense sleeping sickness outbreak in eastern Uganda
    Fèvre, EM
    Coleman, PG
    Odiit, M
    Magona, JW
    Welburn, SC
    Woolhouse, MEJ
    [J]. LANCET, 2001, 358 (9282) : 625 - 628
  • [6] The human serum resistance associated gene is ubiquitous and conserved in Trypanosoma brucei rhodesiense throughout East Africa
    Gibson, Wendy
    Backhouse, Toby
    Griffiths, Andrew
    [J]. INFECTION GENETICS AND EVOLUTION, 2002, 1 (03) : 207 - 214
  • [7] Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method
    Ikadai, H
    Tanaka, H
    Shibahara, N
    Matsuu, A
    Uechi, M
    Itoh, N
    Oshiro, S
    Kudo, N
    Igarashi, I
    Oyamada, T
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 2004, 42 (06) : 2465 - 2469
  • [8] Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M-avium, and M-intracellulare in sputum samples
    Iwamoto, T
    Sonobe, T
    Hayashi, K
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 2003, 41 (06) : 2616 - 2622
  • [9] Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances
    Kaneko, Hisatoshi
    Kawana, Takashi
    Fukushima, Eiko
    Suzutani, Tatsuo
    [J]. JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS, 2007, 70 (03): : 499 - 501
  • [10] Loop-mediated isothermal amplification for detection of African trypanosomes
    Kuboki, N
    Inoue, N
    Sakurai, T
    Di Cello, F
    Grab, DJ
    Suzuki, H
    Sugimoto, C
    Igarashi, I
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 2003, 41 (12) : 5517 - 5524