Use Of Specificness Primer R2-S2 Have Accredit Sporotrichosis Of Animal Model Paraffin Section And Clinical Sample

Sporothrix schenckii is the causative agent of sporotrichosis, which affects primarily the skin, subcutaneous tissue, lymph vessels, lymphnodes and rarely, visceral organs. Sporotrichosis spread widely in the world, Primary disease in our country northeast fairly pilosity ,on prevail region batch to happen, harm persons heath. Now,this disease diagnose difficulty. Raising the fungus,waste lots of time(about one week), as well masccline rate low; tissue pathology is nonspecific granulomatous inflammation, in telaetiology diagnosis still to remain restrict at HE and PAS dyeing, but because fungus are few, acquiring routine histology method is difficult. For the past few years, scholars have versatile explore on the this diseases'investigation, including serology detection, IMFA, immunopathogenesis and electron microscopy. Because serology detection specific problem fail to continue, although IMFA and immunopathogenesis raise dignosis'speed, antibody standard is difficult. Electron microscopy manipulate multiplicity, eyesight is little, the fungus are difficult to be finded, so to spread is impossible. We subject class have genomic DNA was extracted from 50 strain of S.schenckii, including 49 clinical isolates and 1 referrence strain,as well as from Mucor, Aspergillus fumigatus and Canada albicans, respectively, as controls. Four primers were designed according to previous studies(the primer SS3-SS4, S2-R2, SSHF31-SSHR97 and ITS3-SSP). So far, the primer S2-R2 (target the chitin synthase 1 gene) of S.schenckii highly specific and sensitive.Objective: The research will depend sporotrichosis of animal model as well to research sample, further screen four primers ,get the primer that specific and sensetive, use it detect paraffin imbedding tissue and clinical samples'sporotrichosis, for exactly, rapid diagnosis sporotrichosis in the gene level, providing based on the therapy as early as possible, decreased misdignosis and loss.Materials and methed: 1.12 mice trabunt fractionate two sets, experiment group animal inject sporotrichosis suspension intradermally, as well as different time remain skin slowedpulse tissues, discerning to run eumycete cultivation and extract DNA that skin slowedpulse tissues, have the PCR amplification. To compare different primers'sensitivity and specificness. 2. Take 30 samples dalian and changchun area clinical doubtful sporotrichosis, tissue pathology show granulomatous inflammation paraffin section preparatio, introduction improved microwave deparaffinage, LN grind-CTAB break- wall method extract DNA, we use the primer S2-R2 targeting the chitin synthase 1 gene is the specific and sensetive primer for S.schenckii. using specificity primer have fungus DNA PCR amplification reaction, taking PCR amplification production have AGE, using UV spectrophotometer observation. Analysis electrophoresis, identify diagnosis sample whether or not sporotrichosis. 3. Get 10 samples clinical doubtful sporotrichosis acromegalic skin-slowed pulse 0.4×0.8CM2 , to divide equally group discern use for traditional mycology appraisement (fungus train, tissue patholgy) PCR detection. Skin skin-slowed pulse have dull knife, removel dopant and then homogenate, centrifugate to get underlayer sediment, microamount extractio (LN-grind CTAB method) extractio fungus DNA, using specificity primer S2-R2 have fungus DNA PCR amplification reaction, taking PCR amplification production have AGE, using UV spectrophotometer observation.Result:1. Four pairs of primers S2-R2,SSHF31-SSHR97,ITS3-SSP,SS3-SS4 all could be amplification objective straps in the rat skin of sporotrichosis animal model, however primer SSHF31-SSHR97 need higher density, with in vitro cultrue condition have the same results, primer S2-R2 are the most sensitive and specific primer.2. 30 samples preliminary diagnosis sporotrichotic paraffin samples amplification masccline straps are 22 samples, masccline rate is 73.33%. The result of eumycete cultrue is 22 samples. 20 samples PCR have the masccline straps in 22 eumycete cultrue samples, masccline is 91%. In 8 eumycete culture samples are negative, there are 2 samples PCR have masccline straps, masccline rate is 25%. 7 samples PCR have masccline straps in the 10 samples of chuangchun area, masccline is 70%. Each 1 sample Mucor, Aspergillus fumigatus and albicans of PCR amplification is negative.3. 10 samples clinical doubtful sporotrichosis acromegalic tissue get from trunk (5 samples), wrist (3 samples), prosopo (1 sample), eyelid (1 sample). Fresh tissue eumycete culture 9 samples are masccline. Colony 3-10 days come out. 10 samples get out genome DNA, using primer S2-R2 run PCR get 9 samples masccline, with eumycete culture has the same result.Conclusion:1.Using sporotrichosis infected mouse animal model to study and research samples acquired the most specific, sensetive primer also to aim directly at chitin synthase gene 1 S2-R2, with the sporotrihosis pure culture strain have the same consequence.2.Comparing to the traditonal method, improved microwave deparaffinage, LN grind-CTAB breaking-wall method could get enough, high-quality DNA, accordingly raise PCR reactive masccline rate.3.To make use of specificity S2-R2 primer detect paraffin section sporotrichosis masccline rate is 91%. If culture negative essays also culd be detected, masccline rate is 25%. The method is not only utilize lab preservative lots of paraffin section have scientific research and clinical retrospective diagnosis, but also could have stubborn diseases'diagnosis and antidiastole.4.To make use of specificity S2-R2 primer detect clinical fresh biopsy tissue sporotrichosis, in accordance with eumycete culture, masccline rate is 100%. The method has manipulate simplely, save time, sensetive and specificity highly, accurate reliability etc, refer to sporotrchosis rapid diagnosis.