Study On Gene Chip For The Detection Of Common Pathogens Causing Fungal Keratitis

Fungal keratitis is a kind of serious ophthalmopathy to induce blindness. It is theimportant prerequisite of curing this ill and reducing the incidence of blinding todetect pathogens efficiently. For a long time, the conventional identification ofpathogenic fungi in clinical microbiology laboratories based on phenotypic featuresand physiological tests such as direct smear and fungi culture, which is not onlytime-consuming but also lowly sensitive. Therefore, it is the most important step inclinical diagnosis to establish detection of a fungal pathogen to the species level withhigher specificity, sensitivity and accuracy. It was the aim of our study to develop aspecies identification gene chip by making use of the universal primer andoligonucleotide probes in the field of molecular detection for identification ofcommon cornea fungal pathogens. An economically more efficient approach would bethe application of a protocol which is capable of identifying a panel of relevant speciesin a highly parallel fashion.The main points of the results from this study are summarized below:1. Extracted 12 fungal DNA with guanidinium isothiocyanate method. Universalprimers were designed and synthesized. 12 fungal DNA were amplified in a 30μlmixture of 20×buffer 1.5μl, MgCl₂ 2.0mM, dNTP 200μM, Taq DNA polymerase 2U,template DNA 3μl and universal primer 8pmol, add ddH₂O to 30μl. Cycling includeddenaturation for 45s at 94℃followed by annealing for 60s at 55℃, extension for 60sat 72℃and cycling was concluded with a final elongation for 5 min at 72℃. PCRproducts from amplications with universal primer were run on agarose gel show that550-650bp DNA fragments were amplified from all the 12 fungal strains. We evaluated a new PCR technique for determination of pathogenic fungal by usinguniversal primer.2. Using bacterial or virus and human DNA detected the specificity of PCR. No bandwas observed in ethidium bromide-stained gel when human and bacterial or virus wasused as template. It indicated that this method was very specific to detect fungalspecies. Serial 10-fold dilutions methods were used to test the sensitivity of PCR.Sensitivity test showed that this technique can detect a band in agarose gel at 10 fgDNA. The result of serial dilutions test indicated that this PCR technique also has ahigher sensitivity for detection of fungal species.3. Glass slides were modified with aldehyde group in order to develop gene chips. 12probes were designed and synthesized aimed at fungal species, such as Fusariumsolani, Fusarium moniliforme, Fusarium poae, Fusarium oxysporum, Aspergillusfumigatus, Aspergillus flavus, Aspergillus niger, Aspergitlus terreus, Penicilliumimplicatum, Curvularia lunatus, Alternaria alternate and Candida albicans respectively.Every probe was modified by amino-group and then was spotted on glass slides. PCRproducts were hybridized to the gene chip in an optimizing condition of reversehybridization. The optimizing hybridization temperature was 42℃, lasting for 1h,washing for 1min. Hybridization of PCR products from 12 fungal strains showed goodfluorescence signals in their site. So we developed a fluorescent labelling gene chiptechnique for the identification of common pathogens causing fungal keratitis.4. PCR products from human or other microorganism DNA and the mixture of severalfungal strains, such as Fusarium solani, Aspergillus fumigatus, Penicillium implicatumand Curvularia lunatus, hybridized to gene chip in order to test the specificity. PCRproducts from the mixture of several fungal strains also hybridized well to genechip,no cross-reaction signal appeared. PCR products from human or other microorganismhybridized to genechip appeared no signal.5. Serial 10-fold dilutions methods were used to test the sensitivity of hybridization ingene chip. Serial 10-fold dilutions methods showed that the sensitivity of gene chip is1pg DNA. PCR products from Fusarium solani and Aspergillus fumigatus hybridizedto gene chip appeared fluorescence signals at 10^-1 dilution, but showed no band in agarose gel.6. Selecting some gene chip from different batches hybridized to PCR product ofFusarium solani to detect the coefficient of variation appraising repeatability of genechip. Scanning aldehyde group glass slide by atomic force microscopy study thesurface shape. Using gene chips preserved for different time to hybridize observingthe timeliness. The coefficient of variation of gene chip from different batcheshybridized to PCR product is little, showed well repeatability. Scanning aldehydegroup glass slide by atomic force microscopy showed the chemical changes in genechip. Using gene chips preserved for 2 months to hybridize observed fluorescencesignals. The aldehyde group gene chip can detect common pathogens causing fungalkeratitis rapidly and sensitively.7. The species-specific oligonucleotide probes to identify 12 fungi species were addedhomopoly tails and then inoculated on a nylon membrane. Fungi DNA was amplifiedby PCR with fungi-universal primers labeled with biotin. The PCR products wereidentified by hybridization with the species-specific probe fixed on the nylonmembrane. Alkali phosphatase colouring method was used to detect the hybridizationproducts. All 12 standard fungal strains were amplified by PCR, 12 species-specificprobes only hybridized with its target molecules, and the test manifested these probeswere high specific. We established the method of using PCR combining with reversedot blot hybridization to produce visual gene chips for common pathogenic fungal incornea. Using PCR with reverse dot blot hybridization to prepare visual gene chipscan identify the common pathogenic fungi in cornea, which is a simple, rapid andaccurate method that may have a broad prospect for clinical application.8. Used slide or nylon membrane gene chip test 82 culture isolates, and used slide ornylon membrane gene chip test 170 clinical samples. The outcome of gene chip testwas compared with smear and cultivation. In 82 culture isolates, the positive numbersof slide gene chip test were 73, and that of nylon membrane gene chip test were 69.There is no significant difference between these two methods. In 170 clinical samples,the positive numbers of slide gene chip test were 147, that of nylon membrane genechip test were 143, and that of culture method were 153, so there is no significant difference between these three methods. However, the positive numbers of KOHsmear were 113, so significant difference was found in the sensitivity between genechip test and smear. The sensitivity of gene chip was higher than that of KOH smear intesting clinical samples, which was completely consistent with fungal culture. Genechip carried out detection for clinical diagnosis, compared with KOH smear andculture method, appraised its clinical application value preliminarily.This study confirmed that the application of gene chip technique for the detectionof fungal keratitis is entirely feasible with an ideal specificity, accuracy and sensitivity.Through PCR and hybridization, gene chip can complete detection and identificationof the most common species of pathogen causing fungal keratitis in 3-4h. Thetechnique is easy to operate in practice with a broad prospect for clinical application.