弓形蟲與異尖線蟲的免疫診斷和分子檢測(cè)

1、弓形蟲與異尖線蟲的免疫診斷和分子檢測(cè)March, 20112022-3-232022-3-23LAMP developed in parasites study (65/434,14.97%) March, 2011No reports about Anisakis detection by LAMP2022-3-232022-3-23Background Approximately one third of worlds population is infected with T. gondii , and the infection rate is 12.3% in China. Ther

2、e are numbers of different assays to detect T. gondii, including Sabin-Feldman dye test, direct agglutination test (DAT), indirect fluorescent antibody test (IFAT), ELISA and PCR. In the past decade, the use of the PCR has made a significant improvement in both the prenatal diagnosis of congenital t

3、oxoplasmosis and the detection of acute disease in the immunocompromised patient, especially for nested PCR and real-Time PCR. LAMP is more simple, sensitive and specific than these methods and no need for expensive equipment and long reaction time.2022-3-23MethodThe LAMP assay based on B1 gene of T

4、. gondii was developed at 65 for 1h with genomic DNA of T. gondii and other parasites. Six weeks old BALB/c mouse were infected by intraperitoneal injection of 20 virulent tachyzoites of the T. gondii RH strain. The blood samples were collected at 1, 3 and 5 dpi for T. gondii detection by LAMP. 2022

5、-3-232022-3-23Sensitivity of LAMP using the extracted DNA from T. gondii tachyzoiteGenomic DNA 100 pg 10 fg(B) LAMPM, DL2000 markerN, negative control1, 1 ng2, 100 pg 3, 10 pg4, 1 pg 5, 100 fg 6, 10 fg7, 1 fg(A) Nested PCR 2022-3-23SpecificityN, Negative control1, Toxoplasma gondii2, Trypanosoma eva

6、nsi3, Paragonimus westermani 4, Clonorchis sinens5, Angiostrongylus cantonens 2022-3-232022-3-23Early Detection of T. gondii Infection in mice The assay was able to detect T. gondii DNA in all mice blood samples at 1 dpi, which demonstrated that LAMP was effective for early diagnosis.2022-3-232022-3

7、-23Conclusions (1) Establishes the LAMP assay based on B1 gene for sensitive, specific and early diagnosis of T. gondii;(2) The detection limit of LAMP assay is 10 fg of T. gondii genomic DNA and no cross-reaction with the DNA of other parasites;(3) Provides the first report that the LAMP assay is a

8、n early diagnostic tool for toxoplasmosis control.2022-3-23II. Anisakis detection by LAMP and ELISAMore than 1 billion people worldwide are infected with one or more species of gastrointestinal nematode worm parasite, which cause a wide range of conditions from the mild to the lethal. And the nemato

9、de worm species most commonly involved in human infections is Anisakis simplex.To date, there are some methods to detect Anisakis, such as endoscopy, immunodiagnostic assays and PCR, but no reports about the LAMP developed to detect the Anisakis infection.Background2022-3-232022-3-23Umehara A, Kawak

10、ami Y, Ooi HK, et al: Molecular identification of Anisakis type I larvae isolated from hairtail fish off the coasts of Taiwan and Japan. Int J Food Microbiol 2010, 143:161-165. Infection rate is 65.2% of marine fish in the area of the east China sea adjacent to land , and no case report in China. 20

11、22-3-232022-3-23We collected some Anisakis from marine fish in the area of the East China Sea and identified them as A. Pegreffii by RFLP (restriction fragment length polymorphism ) method. We construct the rat model by intragastric administration and detected the IgG antibody specific to A. Pegreff

12、ii larvae in rats. The LAMP assay based on ITS2 sequence of Anisakis was developed at 65 for 1h with genomic DNA of A. Pegreffii and the serum of rat model.Method 2022-3-232022-3-232022-3-232022-3-23Anisakis identification by RFLP (A. Simplex, A. Typica, A. Pegreffii, A. Physeteroid, A. Paggiae, A.

13、Brevispiculata, A. Simplex C Nascetti, Pseudoterranova ceticola) M 1 2 3 4 5 M 1 2 3 4 5 ITS COXIIM, DL2000 marker1, negative control2, Hinf3, BfaI4, RsaI 5, AluI2022-3-232022-3-232022-3-23Anisakis identification by RFLPM, DL2000 marker L, 100 bp marker S, A. Simplex P, A. Pegreffii SP, Recombinant

14、genotype1, negative control 2, HinfI 3, BfaI 4, RsaI 5, AluIM 1 2 3 4 5ITSCOXIIM 1 2 3 4 5L S P SPITS (HinfI)500bp2022-3-23Anisakis detection by LAMPM, DL2000 markerN, negative control1, 100 ng 2, 10 ng3, 1 ng 4, 100 pg 5, 10 pg6, 1 pg2022-3-23Detection of Anisakis infection in ratM, DL2000 marker1,

15、 Negative control2, 3 dpi3, 7 dpi4, 14 dpi5, 28 dpi6, 42 dpi7, 56 dpi8, 70 dpi2022-3-23IgG kinetics against Anisakis in rat with in-direct ELISA ODprimary infection / reinfectionDays2022-3-232022-3-23Physiology and pathology of human with Anisakis infectionAudicana MT, Kennedy MW: Anisakis simplex:

16、from obscure infectious worm to inducer of immune hypersensitivity. Clin Microbiol Rev 2008, 21:360-379, table of contents No serious physiology and pathology be found in rat infected with Anisakis? 2022-3-232022-3-23Kim KH, Eom KS, Park JK: The complete mitochondrial genome of Anisakis simplex (Asc

17、aridida: Nematoda) and phylogenetic implications. Int J Parasitol 2006, 36:319-328 ITS and coxII are the target genes to design primers for detection2022-3-232022-3-23Kim KH, Eom KS, Park JK: The complete mitochondrial genome of Anisakis simplex (Ascaridida: Nematoda) and phylogenetic implications.

18、Int J Parasitol 2006, 36:319-328 We did not gain the LAMP primers based on coxII with software online and the detection limit based on ITS sequence is only 10 pg of Anisakis DNA . How to select the target gene with more sensitivity and design primers should be further studied. 2022-3-23Ideas of prot

19、eomic analysis of Toxoplasma gondii and Schistosoma japonicum2022-3-23March, 20112022-3-23Proteomic analysis of some parasites(346)March, 20112022-3-23Background of T.gondii proteome Model organism for the study of intracellular pathogens Completion of the genomes for T. gondii type I (GT-1) typeII

20、(ME49) type III (VEG) T. gondii global proteome T. gondii subproteome （Secreteome） T. gondii post-translational modifications2022-3-232022-3-23Proteomic analysis of T. gondii proteins regulated during the differentiation process from tachyzoite to bradyzoite stageDynamic unloading and purification o

21、f tachyzoite and bradyzoite during conversionHarvest and purification of tachyzoite and bradyzoite from miceConstruct the model for T. gondii conversion in vitro Construct infection model with T. gondii in vivo2022-3-232022-3-232DE profiles of proteins before and after T. gondii conversionAnalysis of 2DE profilesIdentification of conversion-associated proteinsConfirmation the conversion-associated proteinsGene Ontology analysis Discussion: Construct biological function networ