| Objective: Establishment of animal model of Cryptococcus neoformans bloodstream infection,collecting animal plasma and bronchoalveolar lavage fluid,at the same time FQ-PCR,blood culture,ink stain,histopathology,latex agglutination test control observation,to explore the FQ-PCRdetection system in the animal model of DNA in the samples of diagnosis and comparison of four kinds of method for the optimization of the selected,lay the basic clinical fungal PCR kitdevelopment,to achieve the early clinical diagnosis and treatment of fungal infection,reduce the mortality of clinical fungus infection rate and treatment cost.Methods:1 Experimental study design:(1)the standard strain cultivation and preparation,standard of Cryptococcus neoformans inoculated in PDA flat 37 DEG C after incubation for 48 hours,byinoculating loop scraping colony dissolved in saline into the sterilized EP tube;the blood cell counting plate count,made cell concentration is about suspension 106 CFU /ml,storagesolution as the standard strain,placed in the refrigerator spare-80 C.(2)animal model,32 male SD rats,using randomized divided into 4 groups,at the same time weighing,number,respectively,in captivity,provide food and water on time.Group A: immunosuppressiveinfection group(n=10 only): injection of cyclophosphamide(150mg/kg)solution were immunosuppressed rats,liquid infection animal hanging tail vein injection of Cryptococcus neoformans strains;group B: normal infection group(n=10),saline injected,model is established after tail intravenous injection of Cryptococcus neoformans strains suspensioninfected animal group C: normal control group(n=6): normal diet;D group of immunosuppressive control group(n=6);only the injection of cyclophosphamide(150mg/kg)solution wereimmunosuppressed rats.A+B as the experimental group(third days after infection were as control group(C+D),third days after infection were 4 only)2 The full text of statistical methods in data processing and analysis are used SPSS20.0statistical software for statistical processing,the experimental group and the control group in the acquisition of animal serum and bronchial brain vesicles lavage specimens after FQ-PCR testing results are compared.The experimental group and the control group ?2 test on different days,general first when P<0.05,the experimental group and the control group had significant difference.Each experimental group at different time points and the same control group two two comparison,the level of test to adjust for a--0.05/2(5.1),that is,when P<0.00625,the difference was statistically significant.Results:1 Immunosuppressive infection group 24 hours after infection there began to appear poor appetite,decreased activity,hair loss of luster,rat hair disheveled,her head before the footbody arched by;normal infection group,only 3 cases reduced appetite,activity less,the control group with good general condition;2 Rats were sacrificed third days after infection from brain tissue and other organs(liver,spleen,brain).The experimental group compared with control group of rats brain tissuedensely gray white nodules uplift,slightly larger spleen and other organs showed no abnormalities;3 The plasma 101CFU/ml of normal rats suspension were 1:1,1:2,1:3,1:4 dilution,Candida tropicalis standard strains of 102CFU/ml suspension were 1:1,1:2,1:3,1:4 dilution,DNA extraction for PCR amplification,repeat8 times.The standard strain of Cryptococcus neoformans suspension was diluted 1:1 can fully amplified,and by 1:2,1:3,1:4 diluted sampleno amplification or only partial amplification,sensitivity of the four kinds of reaction system can detect all can reach 105CFU/ml;4 The collected serum and lung tissue extraction of DNA detected by FQ-PCR,the positive rate of the experimental group than in the normalcontrol group and immune group is high,P<O.05,the difference was statistically significant,with the infection when asked to extend,the positive rate gradually increased,infection after 8 hours before the experiment group FQ-PCRresults and immune suppression and normal control group there were no significant difference;5 In the FQ-PCR and blood culture,capsular antigen detection,ink stain four comparison of sensitivity of Cryptococcus neoformans detection,FQ-PCR test the highest sensitivity,the sensitivity was 100%,and the capsular antigen was 96.2%,the sensitivity of blood culture was 58.8%,the lowest,FQ-PCR technology in the sensitivity of the diagnosis of Cryptococcus neoformans was higher than that of blood culture,ink stain,capsular antigen test(?2 = 8.694,P = 0.031);FQ-PCR technology in the specific diagnosis of Cryptococcus neoformans was higher than that of blood culture,ink,capsular antigen test(?2= 12.129,P = 0.021);6 The 101 copy number /ul is out of the lowest detection concentration of FQ-PCR,the results show that: the method of using real-time fluorescence quantitative PCR to detect the use ofthe FQ-PCR sensitivity,the sensitivity was 101 copy number /ul copy number;7 In six strains of Cryptococcus neoformans,only showed obvious specificity amplification,five strains were not found to have residual fluorescence curve growth phenomenon,the above results further illustrate the detection of Cryptococcus neoformans FQ-PCR detection methodhas a good specificity;8 Respectively will taste plasmid standard FQ-PCR detection application in 109 copy number/ul,107 copy number /ul,105 copy number /ul copy number three was defined as the high,medium and low,by examining the repetitive batch,visible above the three class of the CV values were 0.59%,0.90% and1.19%,the results into a step that FQ-PCR method has very good intra assay repeatability in the detection of Cryptococcus neoformans.Conclusion:1 The FQ-PCR detection technology has high for Cryptococcusneoformans sensitivity andspecificity;2 The FQ-PCR method has very good intra assay repeatability in the detection of Cryptococcus neoformans;3 FQ-PCR detection technology can be used for accurate detection,gene diagnosis,and thequantitative range,strong specificity,overcome the false positive. |
PDF Full Text Request