Dissertation > Excellent graduate degree dissertation topics show

Target Sequence Selected and Studied from Retrotransposon DNA of Schistosoma Japonicum for the Diagnosis of Schistosomiasis

Author: GuoJunJie
Tutor: XiaChaoMing
School: Suzhou University
Course: Pathogen Biology
Keywords: Schistosoma japonicum nucleic acid detection retrotransposon target sequence selected
CLC: R532.21
Type: PhD thesis
Year: 2012
Downloads: 67
Quote: 0
Read: Download Dissertation

Abstract


Nucleic acid detection has the same diagnostic value with pathogen diagnosticmethod, and the application of nucleic acid detection assays in the diagnosis ofschistosoma infected and therapy evaluation has become the major study spot inmolecular diagnostic techniques of Schistosomaisis. The choice of target sequences usedfor DNA detection was one of the key factor for determining the sensitivity andspecificity of nucleic acid detection assay. In previous studies done by our group, weidentified a230bp sequence of high sensitivity and specificity from the retrotransposonSjR2of Schistosoma japonicum in detecting schistosoma DNA from infected sera ofrabbit modle and human. In this study, new primers from the25novel retrotransposonswere designed and their sensitivity and specificity for detecting S. japonicum DNA weretested，then，we found a303bp sequence from retrotransposon SjCHGCS19; we alsoexplored the key factors for selecting a new perspective sensitive target DNA sequencefor the diagnosis of schistosomiasis; then, nested-PCR method target this303bpsequence was successfully established and was used to evaluate the utility for earlydetection and therapy evaluation in infection rabbit model. Furthermore，the nested-PCRmethod was to evaluate the ability for detedction of S. japonicum human infection andefficacy of chemotherapy. Meanwhile, we also established a new LAMP assay targetsequence from SjCHGCS19, and the LAMP assay were used to evaluate the ability fordetedction of S. japonicum of rabbit infection.This study includs five parts:Part Ⅰ Target sequence selected from25novel retrotransposonsDNA of Schistosoma japonicum for the diagnosis of schistosomiasisIn the previous done by our group, a230bp target sequence from retrotransposonSjR2showed high sensitivity and specificity for detecting S. japonicum DNA fromserum of infected host based on polymerase chain reaction (PCR) methods. In this study, new primers from25novel retrotransposons were designed, PCR assay was used,and their sensitivity and specificity for detecting S. japonicum DNA were evaluated.Interestingly, a new303-bp sequence from non-LTR retrotransposon SjCHGCS19(amplified segment:2049bp~2334bp) showed high sensitivity and specificity. The PCRassay detected as little as21.5fg/μL of S. japonicum DNA.The excepted PCR productwas amplified from both S. japonicum and S. mansoni, but no cross-reaction wasdetected with DNA samples representing of C. sinensis, P. Westermani and T. spirals.The sensitivity and specificity of other retrotransposons were different respectively.Part Ⅱ Study on25novel retrotransposons target sequence ofdetecting S. japonicum DNA by bioinformaticsTo clarify the correlated factor with the sensitivity and specificity of detecting S.japonicum DNA, we studied on25novel retrotransposons and SjR2previous used byour group by bioinformatics. The sensitivity of detecting S. japonicum DNA washighly related to the complete copies of these retrotransposons and the activity of ESTs,based on the multiple linear regression analyze, and also related to the proportion ingenome. Interestly, the230bp from SjR2and303bp from SjCHGCS19both have thehigh complete copies(respectively400,793), higher partical copies (respectively23,755,17,373) and higher activity of ESTs(respectively79,39),and they are both blong to theSR2group of non-LTR retrotransposon. We conclude that one of the key point factorof effecting the sensitivity of detecting S. japonicum DNA based on PCR assay was thechoice of target sequence, the complete copies and activity of ESTs of the targetsequence. The specificity of PCR assay target these retrotransposon was related to thehomology of target sequence with other species.Part Ⅲ The application of303bp target sequence fromSjCHGCS19based on nested-PCR assay was applied in early diagnosisin rabbit infection of S. japonicum and therapy evaluated.Primer were designed target the303bp sequence from SjCHGCS19, thesensitivity and specificity of detecting of S. japonicum DNA were studied based onnested-PCR, and the early diagnose and therapy were evaluated by detecting the serum samples of different intensive infection rabbit model. To determine the limit of the303bp DNA fragment for detecting S. japonicum DNA,10-fold serial dilutions ofstandard plasmid clones of SjCHGCS19were amplified by nested-PCR assay. Theminimum detectable amount of standard plasmid from SjCHGCS19was only2.02copies, whereas the230-bp fragment from SjR2was10.2copies(having notpublished,done by our group). In order to observe the stability of this303bp targetsequence based on nested-PCR assay, different issue from rabbit infected with S.japonicum including female,male adult worms, liver homogenate and sera of rabbitpost infection were detected. The results showed that the303bp specific DNA fragmentof S. japonicum was amplified by nested-PCR assay when the different samples DNAwas used as templates. The specificity result of this303bp sequence based onnested-PCR showed the amplified band both appeared from S. japonicum and S.mansoni, but no cross-reaction was detected with DNA samples representing of C.sinensis, P. Westermani and T. spirals. As to the early diagnosis and therapyevaluation, this303bp sequence from SjCHGCS19could detect the specific DNA at the3dstpost-infection, and then the DNA in serum became negative at the18thweekpost-treatment. These data were1week later than the17thweek post-treatment of230bp sequence from SjR2based on nested-PCR done by our group. So,we concludethat targeting303bp sequence from SjCHGCS19based on nested-PCR assay may beuseful to early-infection for diagnosis S. japonicum DNA and for therapy treatmentevaluated.Part Ⅳ The application of303bp target sequence fromSjCHGCS19based on nested-PCR assay in human schistosomiasisdiagnosis and therapy evaluatedOn the base of the encouraging results from animal model, target this303bpsequence from SjCHGCS19based on nested-PCR was employied to detect43casessera of patients and51health human when the parasitologic examination was used asthe reference test. The results of nested-PCR detection showed that the a sensitivity of97.7%and a specificity of96.1%. The positive predictive values was97.7%, thenegative predictive value was96.1%。Therapy evaluation on47cases of clinical patientes(their EPG all were positive) from3,6and9months after praziquantel. Seraof47cases from3,6and9months after therapy, the negative rate was36.2%,89.4%and87.2%,respectively,based on nested-PCR, it showed a clear upwardtrend,especially the negative rate reached89.4%on6months after therapy,showinggood therapy evaluation. while the negative rate of IHA was23.4%,42.6%and31.9%respectively; the negative rate of ELISA was19.1%,17.0%and25.5%respectively.Part Ⅴ A new LAMP assay target sequence from SjCHGCS19was choiced and LAMP method was evaluatedAt the base of the encouraging results of the nested-PCR targeting the303bpsequence from SjCHGCS19retrotransposon, we tried to find target sequence from theSjCHGCS19retrotransposon for loop-mediated isothermal amplification (LAMP)reaction, a sensitive, simple and rapid technique. Four different target were selected andprimers were designed,the sensitivity and specificity was evaluated by detecting adultworm, then we found a target sequence(SjCHGCS19-2, amplified position located inthe211bp~441bp) suitable for LAMP assay. The sensitivity of detecting S. japonicumDNA of adult worm based on LAMP assay targeting SjCHGCS19-2was130fg/μL, itwas lower than303bp from SjCHGCS19based on nested-PCR(21.5fg/μL)and230bpfrom SjR2based on LAMP methods(0.08fg/μL). and there were cross-reaction with S.japonicum and S. mansoni, but no cross-reaction with C. sinensis, P. Westermani andT. spirals. These result further proved that the choice of target sequence play a key rolein determining the sensitivity of LAMP assay.

Related Dissertations

  1. Purification of Recombinant Schistosoma Japonicum Protein SjGST and Preparation and Characterization of the Monoclonal Antibody,R392
  2. The Preliminary Study of the pcDNA3.1(+)-SjP14 Nanometer Microballoon-DNA Vaccines,R392.1
  3. Construction of Prokaryotic Shuttle Expression Plasmid pBCG-Sj14-Sj26 and Identify Their Expression in Mycobacterium Smegmatis.,Q78
  4. Study on Pharmacokinetics and Spatial and Temporal Expression in Mice of DNA Vaccine against Schistosoma Japonicum,S855.91
  5. Establishment of Pichia Pastoris and Transgenic Medicago Sativa Expression System for Schistosoma Japonicum Antigen Sj23,Q943.2
  6. Prokaryotic Expression and Purification of Schistosoma Japonicum 23KD Membranin in E. Coli BL-21(DE3) and Comparative Study on the Anti-infective Effect between Bivalent Membrane-anchored and Secreted Expression Vaccine,R392
  7. Detection of Shrimp White Spot Syndrome Virus by Loop-mediated Isothermal Amplification Combined with a Lateral Flow Dipstick,S945.46
  8. Cloning, Expression and Biological Function Analysis of Schistosoma Japonicum Frizzled Family Receptors,S852.7
  9. Development of Molecular Fingerprints Based on Retrotransposon and Applications in Rice Breeding,S511
  10. Studies on the SJCHGC06868 Protein of Schistosoma Japonicum,R383.2
  11. Schistosoma japonicum eggs on TNBS -induced colitis in mice models of regulatory T cells and B cells regulate the impact of,R574.62
  12. Establishment of the Inter-retrotransposon Amplified Polymorphism (IRAP) Marker System and Identification of Apple Bud Mutation Clones,S661.1
  13. The Expression and Implication of AKT1, Survivin, caspase-3 in Colorectal Cancer with Schistosomiasis,R735.34
  14. Epidemiological Surveillance and Molecular Analysis for Three Neurotropic Virus Infection in Western Region of China,R742.9
  15. Functional Identification of Hepatic Stellate Cells from Mice Infected with Schistosoma Japonicum,R532.21
  16. Modelling Eggs Distribution and Immune System Dynamics in Schistosome Japonicum Infected Hosts,R532.21
  17. Studies on Two Isoforms of Adenine Phosphoribosyltransferase (APRT) of Schistosoma Japonicum,R392
  18. Study on the Effect and Mechanism of Activated Retrotransposons for the Adaptive Ability in Candida Albicans,R379
  19. The Analysis of the Interaction of the 23kDa Integral Membrane Protein of Schistosoma Japonicum and Human Immunoglobulins,R392
  20. Characterization of T-cell Immunogenicity of Prosaposin Protein of Schistosoma Japonicum,R392
  21. Screening and Identification of Th Cell Epitopes from Schistosomula Antigen of Schistosoma Japonicum,R392

CLC: > Medicine, health > Internal Medicine > Parasitic diseases > Helminthiasis > Trematodes > Schistosomiasis
© 2012 www.DissertationTopic.Net  Mobile