title: Establishment and application of a loop-mediated isothermal amplification method for simple, specific, sensitive and rapid detection of Toxoplasma gondii.
abstract: The Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high simply, specificity, sensitivity and rapidity. In this study, A LAMP assay with 6 primers targeting a highly conserved region of the GRA1 gene was developed to diagnose Toxoplasma gondii. The reaction time of the LAMP assay was shortened to 30 min after optimizing the reaction system. The LAMP assay was found to be highly specific and stable. The detection limit of the LAMP assay was 10 copies, the same as that of the conventional PCR. We used the LAMP assay to develop a real-time fluorogenic protocol to quantitate T. gondii DNA and generated a log-linear regression plot by plotting the time-to-threshold values against genomic equivalent copies. Furthermore, the LAMP assay was applied to detect T. gondii DNA in 423 blood samples and 380 lymph node samples from 10 pig farms, and positive results were obtained for 7.8% and 8.2% of samples, respectively. The results showed that the LAMP method is slightly more sensitive than conventional PCR (6.1% and 7.6%). Positive samples obtained from 6 pig farms. The LAMP assay established in this study resulted in simple, specific, sensitive and rapid detection of T. gondii DNA and is expected to play an important role in clinical detection of T. gondii.
authors: Jilin Academy of Animal Husbandry and Veterinary Medicine, Changchun, Jilin 130062, China.
authors: Lili
authors: L
authors: Cao
authors: Ronghua
authors: R
authors: Cheng
authors: Lin
authors: L
authors: Yao
authors: Shuxian
authors: S
authors: Yuan
authors: Xinhua
authors: X
authors: Yao
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D057230:Limit of Detection; D051379:Mice; D008807:Mice, Inbred BALB C; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000469:parasitology*; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*; D014124:Toxoplasmosis, Animal / Q000097:blood / Q000469:parasitology*; D015047:Zoonoses / Q000469:parasitology*
chemical_list: D000953:Antigens, Protozoan; D016054:DNA, Protozoan; C412913:GRA1 antigen
references: Nucleic Acids Res. 2000 Jun 15;28(12):E63
references: 10871386
references: J Parasitol. 2011 Apr;97(2):262-4
references: 21506784
references: Vet Parasitol. 2012 Jun 8;187(1-2):53-6
references: 22265079
references: Science. 1988 Jan 29;239(4839):487-91
references: 2448875
references: J Clin Microbiol. 1996 Nov;34(11):2850-2
references: 8897199
references: Am J Obstet Gynecol. 2005 Feb;192(2):564-71
references: 15696004
references: Clin Vaccine Immunol. 2012 Jun;19(6):865-74
references: 22496494
references: APMIS. 2010 Aug;118(8):529-42
references: 20666734
references: FEMS Microbiol Lett. 2004 May 1;234(1):93-7
references: 15109725
references: Mol Cell Probes. 2002 Jun;16(3):223-9
references: 12144774
references: J Clin Microbiol. 2000 Nov;38(11):4121-5
references: 11060078
references: Exp Parasitol. 2008 Jan;118(1):75-9
references: 17904554
references: J Clin Microbiol. 2005 Apr;43(4):1625-31
references: 15814976
references: Exp Parasitol. 2009 May;122(1):47-50
references: 19545521
references: Rev Med Virol. 2008 Nov-Dec;18(6):407-21
references: 18716992
references: J Clin Microbiol. 2003 Dec;41(12):5517-24
references: 14662933
references: Parasit Vectors. 2011 Aug 24;4:165
references: 21864327
references: FEMS Microbiol Lett. 2004 May 1;234(1):19-25
references: 15109715
references: J Clin Microbiol. 2001 Jun;39(6):2065-71
references: 11376036
references: Exp Parasitol. 2006 Nov;114(3):220-7
references: 16707125
references: Clin Microbiol Infect. 2002 Oct;8(10):634-40
references: 12390281
references: Vet Parasitol. 2012 Apr 30;185(2-4):296-300
references: 22051073
references: N Engl J Med. 1992 Dec 3;327(23):1643-8
references: 1359410
references: Appl Environ Microbiol. 2003 Aug;69(8):5023-8
references: 12902306
references: Vet Parasitol. 2012 Aug 13;188(1-2):14-8
references: 22483557
references: Parasitol Res. 2007 Jan;100(2):333-7
references: 16896649
references: J Clin Virol. 2008 Apr;41(4):270-6
references: 18296109