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1、Mo l e c u l a r D i a g n o s i s o f Gr a y Mo u l d P a t h o g e n On F r u i t s A n d V e g e t a b l e s Yun-Feng Hu12Jing Cui12 Yan-Ping Wang1Hong-Lian Guo1 Ying Zuo1 Ke Zhang1Wei-Wei Liu1 1Department of Food Science, 2Food nutrition and safe key laboratory ,Institute of Food Engineering and
2、 Biotechnology.Tianjin University of Science &Technology.Tianjin 300457, China. A b s t r a c t Molecular methods used directly on clinical material play an important role in the diagnosis and gray mould pathogen. Molecular diagnostic tests that allow timely and accurate detection of pathogen are al
3、ready implemented in many laboratories. The intent of this review is that the reader will better understand the variety of technical approaches to the molecular genetic testing for the gray mould pathogen. The early molecular diagnosis of gray mould pathogen should significantly aid in the developme
4、nt of therapies tailored to this clinically and pathogenetically distinctive subgroup of bio-prevention. It is therefore particularly timely and appropriate to analyze these issues in detail, specially the extent to which molecular biology has revised traditional diagnostic criteria. The role of gen
5、etic testing in assessing prognosis and identifying high-risk subgroups and in defining basic disease mechanisms and pathophysiology is, however, largely beyond the scope of this scientific statement. Ke y wo r d s : Gray mould pathogen, Specific primers, Diagnostic, Bio-prevention, Pathopoiesia mec
6、hanisms I. INTRODUCTION Gray mould pathogen is one of the leading infectious diseases on fruits and vegetables and is responsible for new cases annually.Botrytis is one of the main diseases in China. Great loss happened while this disease occurred severely, which apparently lowering the yield and qu
7、lity of the crops. It is necessary to strengthen research of rapid, accurate diagnostic techniques The purpose of this review is to highlight the underlying principles in miniaturization, the strategies developed for bioanalysis, and the potential impact on the practice of this rapidly growing medic
8、al discipline. During the past several years, many molecular methods have been developed for direct detection, species identification and susceptibility testing of gray mould pathogen. These methods can potentially reduce the diagnostic time from weeks to days. Currently,PCR-based sequencing has bec
9、ome commonly used to identify many mould species. DNA probes have been widely used for species determination of the most commonly encountered gray mould pathogen. High-density oligonucleotide arrays (DNA microarrays) also have been applied to simultaneous species identification 1and detection of gra
10、y mould pathogen that confers resistance in mould. This review is descriptive and intended to outline the technologic and methodologic approaches to the creation of an integrated genetic analysis instrument based on gray mould pathogen. The review draws on published scientific evaluations of these d
11、evices without regarding to the companies involved in their development. Currently, no molecular tests are routinely available for the diagnosis of gray mould pathogen.Gene expression profiling is on the horizon as a means to improve subclassification of fruits and vegetables-related diseases and to
12、 predict response to therapy. We undertook a gene expression profiling study of gray mould pathogen to establish a molecular diagnosis of this disease. We identified a gene expression signature of gray mould pathogen that distinguished this subgroup from other pathogen and showed that gray mould pat
13、hogen have distinctive clinical features and a favorable overall survival rate after therapy. II. MATERIALS AND METHODS A.Origin and maintenance of pathogen isolate. The isolate of B. cinerea used in all experiments was obtained from an infected pear in a commercial greenhouse in China. The isolate
14、was grown on potato dextrose agar (PDA) and stored as mycelium on 9cm-diameter petri plates at 4C. Mycelial plugs from storage were used to start culture needed to conduct 978-1-4244-4713-8/10/$25.00 2010 IEEEexperiments.Hypha of pear transported immediately to the laboratory. Before use, the fruits
15、 were cleaned with 0.5% NaCl for 5 min and then washed in sterile-distilled water. After air drying, grapes were surface-sterilized by wiping them with a 70% ethanol solution. B.Analysis of Gene Expression and Clinical Data. Through methods described elsewhere, gray mould pathogen was isolated from
16、the fruits and vegetables.Pretreatment gray mould pathogen samples were studied according to a protocol approved by the NCI Institutional Review Board 2. The Bayesian statistical procedure used to create the gene expression-based gray mould pathogen predictor has been described C. Determination of I
17、ntronic Sequences The presence of different primers that could confound PCR-based genotyping methods and the absence of data on the genomic structure of the gray mould gene led us to initially use PCR primers that were complementary to sequences to amplify genomic DNA by Expand PCR (Boehringer Mannh
18、eim, Indianapolis, Indiana) and thereby identify sequences in the gray mould pathogen gene. The final volume for all PCR assays was 50 micro L. Through use of 0.2g of hypha genomic DNA as a target, PCR was done with primersA(5-CGATCGATAGATATGTCAGA-3) and B (5-ATGGACTGTACTTCGTGGAC-3) in buffer D (Inv
19、itrogen, San Diego, California). The buffer contained Tris hydrochloride (pH 8.5), 60 mmol/L; ammonium sulfate, 15 mmol/L; and magnesium chloride, 3.5 mmol/L 3. The primers had been developed to amplify a fragment of genomic DNA (which included nucleotide 460) for detection of the fragment of P729 m
20、utation. The concentration of each oligonucleotide was 0.1 OU/mL (about 0.5 mol/L), and 0.2 L Taq polymerase (Perkin Elmer Cetus, Norwalk, Connecticut) was used. With a Hybaid OmniGene thermocycler (Woodbridge, New Jersey), amplification was done for 30 cycles consisting of denaturation at 94 C for
21、1 minute, annealing at 55 C for 2 minutes, and extension at 72 C for 1 minute. A final extension step at 72 C for 7 minutes was also done. For the initial cycle, 5 L of deoxynucleoside triphosphates (dNTP, 10 mmol/L) was added after the temperature reached 80 C (following the hot start protocol). An
22、 amplified fragment of P729 base pairs was anticipated in the absence of intron sequences 4; the resulting fragment of P729 base pairs showed the presence of an intervening intron. Through a similar strategy, Expand PCR was used to amplify intron sequences that flanked the exons containing the P729
23、. The resulting intron-containing fragments were directly cloned into the plasmid PCR-II; the plasmid was purified and arrayed as described above. These sequences permitted the development of intron-specific PCR primers P729 for the detection of gray mould pathogen. D. Detection of gray mould pathog
24、en by Polymerase Chain Reaction We used PCR amplification to determine whether the P729 transversion was present at the locus. Unpurified PCR products were analyzed by electrophoresis in 2.5% MetaPhor gels (MetaPhor Agarose, FMC Bioproducts, Rockland, Maine) stained with ethidium bromide 5. A DNA fr
25、agment was amplified with P729 primers when it was present, whereas a DNA fragment was amplified with P729 primers when wild-type was present. E. Expression of gray mould pathogen Genes in Primary Molecular Diagnosis. It has been evident, even from the initial descriptions of the disease, that patho
26、gen is usually inherited as a mendelian autosomal dominant trait. Over the last decade, molecular studies using linkage analysis have mapped a number of genetic loci responsible for gray mould pathogen and in the process have provided insights into the considerable clinical heterogeneity characteris
27、tic of this disorder. This genetic diversity is further compounded by intragenic heterogeneity, with a total of more than 100 individual disease-causing mutations identified for these genes. Hence, it is apparent that the precise molecular defect responsible for pathogen usually proves to be differe
28、nt in unrelated individuals. Although several disease-causing mutations have been defined for gray mould pathogen, the clinical consequences of these gene defects and their contribution to disease incidence are not completely understood at present. III. RESULTS A.Development of Gray Mould Pathogen F
29、ig. 3-1 The Electrophoretograms of Phenol-Chloroform extraction We isolated no Gray mould pathogen from pear surface at different growth phase. The isolation results showed that Gray mould pathogen may infect pear fruits and make them rot by wounds in transportation and storage. To discover a gene e
30、xpression signature of gray mould pathogen, we first identified P729 fragment the diagnosis of pathogen was entertained 6. Since the clinical diagnosis of gray mould pathogen is imprecise, we anticipated that this plant disease would be heterogeneous, including some cases of true mould pathogen and
31、other cases with standard pathogen that happened to involve the bio-prevention. We next developed a gene expression-based method to distinguish the gray mould core cases from other ways.Given the heterogeneity of gray mould pathogen, it was challenging to select an optimal set of differentially expr
32、essed genes to be used in the mould pathogen predictor. B. Amplification of P729 fragment of 5 strains of Botrytis cinerea PCR assay to diagnose pear gray mould was established.The sensitivity and specificity were evaluated.The primers P729+/P729- were specific in Botrytis cinerea,and the sensitivit
33、y to diagnostic assay was 0.2g of pathogenic fungi. In addition, the availability of DNA-based diagnosis has led to the identification of increasing numbers of fruits and vegetables with a preclinical diagnosis of gray mould pathogen, usually in the context of genetic testing in selected pedigrees.
34、C. Sequence analysis of the ribosomal ITS region Fig. 3-3 Gel electrophorisis of DNA products of PCR driven by primers ITS1/ ITS4 A single sequence polymorphism in ITS was present between isolates collected from pear . The BLAST similarity search confirmed the results obtained by the species-specifi
35、c PCR analysis, as the ITS sequences obtained from the P729 isolates shared sequence identity with published ITS sequences of P729. Five typical pathogenic strains includingG1, G2, G4, G50, T10, which were isolated from the surface of pear fruits in different areas of the surface of pear fruits, wer
36、e identified as Gray mould pathogen by morphological characters and internal transcribed spacer (ITS) analysis. IV.DISCUSSION A diagnostic test based on gene-expression profiling identified gray mould pathogen that had been verified by an expert panel of hematopathologists. Our study revealed substa
37、ntial difficulty in rendering a reproducible diagnosis of gray mould with the use of current pathological methods. We have used gene expression profiling to establish a molecular diagnosis of gray mould pathogen 7. The pathogen cases identified by our predictor as Molecular Characters have several c
38、linical and molecular features in common with gray mould pathogen identified by M 1 2 3 4 5 5 4 3 2 1 M -2000bp -750bp -500bpcurrent diagnostic methods. A thorough characterization of more such cases will be needed in order to ascertain whether they represent a variant of gray mould pathogen or have
39、 a separate pathogenesis.We propose that the molecular diagnosis of gray mould pathogen by gene expression yields a more precise definition of this pathogen subgroup than current diagnostic methods. Therefore, it appears likely that these cases represent other forms of gray mould pathogen that happe
40、ned to predominantly involve the bio-prevention at presentation.Gene expression profiling identified gray mould pathogen as a subgroup of pathogen with a relatively favorable survival rate after therapy: This example highlights how molecular diagnosis can provide valuable prognostic information that
41、 could help guide the management of gray mould pathogen with diagnostic methods.Despite these striking parallels between gray mould pathogen and other pathogens, there exits important differences.Such cases may indicate that there is a spectrum of diagnostic methods between gray mould pathogen and o
42、ther pathogens, a prospect that can be tested by gene expression profiling and other molecular analysis. In summary, the molecular classifier of gray mould pathogen based on gene expression provides a quantitative and reproducible diagnosis of gray mould that is superior to the best current diagnost
43、ic methods. It could be used to enhance diagnostic accuracy for this curable diseases. Finally, our studies emphasize that gray mould pathogen is a heterogeneous diagnostic category that harbors at least three molecularly and clinically distinct subgroups 8. Clearly, clinical trials in gray mould pa
44、thogen must incorporate gene expression profiling so that these disease entities can be recognized. The molecular diagnosis of these gray mould pathogen subgroups is the first step toward the understanding of pathopoiesia mechanisms that cause these diseases, which will ultimately lead to rational a
45、nd disease-specific treatments. ACKNOWLEDGEMENT The authors are thankful to the professors, Institute of Food and Biotechnology, Tianjin University of Science &Technology, for providing necessary research facilities as well as his keen interest in molecular diagnosis. REFERENCE 1 Barth, T.F., F. Lei
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