| Backgroud & Objective: Varicella zoster virus(VZV)infection can lead to virus-related diseases such as varicella,herpes zoster(HZ)and postherpetic neuralgia(PHN).VZV DNA detection in the virus-related diseases,can serve as a basis for the existence of infection and viremia,as well as judging the degree of nerve injury,disease status and the outcome.At present,the internal detection of VZV DNA in peripheral blood is usually carried out by real-time quantitative polymerase chain reaction(q PCR)detection kit,but the clinical application is limited by a high cost and low sensitivity.Therefore,one of our purposes is to optimize the DNA extraction method,simplify the production of standards,design specific primers,improve the sensitivity,simplicity,rapidity,specificity and cost saving of VZV DNA detection method.Another purpose of this paper is to detect the viral load differences in peripheral blood of VZV-related diseases via our established q PCR method,to use the established q PCR method detect the viral load differences of plasma and peripheral blood mononuclear cells(PBMC)in HZ patients,and to use the enzyme linked immunosorbent assay(ELISA)detect the brain-derived neurotrophic factor(BDNF)and S100 B protein levels in VZV-related diseases.METHOD:Experiment 1st: An optimized PCR for detecting VZV DNA in peripheral blood of HZ patient.1.10 cases of HZ patients were randomly selected,we used boiling method,centrifugal extraction method to extract PBMC DNA,respectively.2.Preparation of standards using nested PCR.3.The q PCR of VZV DNA in PBMCs was carried out by using a VZV Real Time PCR Kit(Liferiver,OD-0024-02),compared with a standard curve constructed in this paper.Experiment 2nd: The clinical application of q PCR method established in this paper and the detection of plasma BDNF,S100 B 1.The analysis of VZV DNA load difference among VZV-related diseases: Samples were collected from 10 cases of varicella,31 cases of HZ,11 cases of PHN and 20 healthy volunteers,and divided into varicella group,HZ group,PHN group and normal group,respectively.The VZV DNA load in the PBMCs was detected by the established q PCR.2.Analysis of differences in VZV DNA Load between PBMC and Plasma before and after treatment: 22 samples were randomly selected from 31 patients with HZ,and the VZV DNA in PBMCs and plasma were measured before and after treatment for 14 days.3.Analysis of the factors for neurological damage assessment in VZV-related disease: The levels of BDNF and S100 B in plasma of varicella group,HZ group and PHN group were detected by enzyme linked immunosorbent assay(ELISA).REUSULT:Experiment 1st: 1.Preparation of the standard: amplified the standard by nested PCR with an size of 385 bp.2.Comparison of extraction methods: Boiling method,centrifugal column method were combined with q PCR method of Liferiver,VZV DNA positive detection rates are 10%,60%,respectively,thus the detection rate of centrifugal column method is higher than the boiling method.3.Comparison of primers: compared with the detection rate of VZV Real Time PCR Kit of Liferiver,the primers designed in the article got a same result of 60%.Therefore,the optimized q PCR method is: VZV DNA was extracted by centrifugal column,standards were produced by nested PCR and primers were designed properly.Experiment 2nd: 1.Comparisons of the positive VZV DNA detection rate and the virus load in peripheral blood PBMC of VZV-related diseases:The positive rate of VZV DNA in PBMC of Varicella group,HZ group and PHN group were 80%,70.97% and 36.36%,respectively.PBMC VZV DNA load in varicella group was higher than that in HZ group and PHN group,and HZ group was higher than PHN group(Z =-2.82,P <0.05;Z =-2.98,P <0.05;Z =-2.47,P <0.05).The VZV DNA detection in normal control group was negative.2.Comparison of positive VZV detection rate and VZV DNA load in HZ group: The positive rate of VZV DNA in PBMC and plasma in pre-treatment 22 patients randomly selected from HZ group were 72.72% and 63.64%,The difference was not statistically significant(?2 = 1.33,df = 1,P> 0.05).The positive detection rate after treatment was 47.06% and 35.29%,respectively.There was no significant difference(?2 = 0.5,df = 1,P> 0.05).VZV DNA load in the PBMC before and after treatment was higher than that in plasma(Z =-4.107,P <0.05;Z =-2.432,P <0.05).The levels of VZV DNA in PBMC and plasma were significantly higher than those before treatment(Z =-4.11,P <0.05;Z =-3.03,P <0.05).3.Comparisons of plasma BDNF in VZV-related diseases: the levels of BDNF in the varicella group,HZ group and PHN group were significantly higher than that in the normal group,and the difference was statistically significant(I-J=190.89,P<0.05;I-J=232.49,P<0.05;I-J=246.65,P<0.05).Compared with HZ group and PHN group,the varicella group had significant lower BDNF content(I-J =-46.60,P <0.05;I-J =-55.75,P <0.05).There was no significant difference between HZ group and PHN group(I-J =-14.15,P> 0.05).4.Comparisons of plasma S100 B in VZV-related diseases: S100 B levels in the varicella group,HZ group and PHN group were significantly higher than that in the normal group,and the difference was statistically significant(I-J=126.55,P<0.05;I-J=164.85,P<0.05;I-J=159.94,P<0.05),varicella group had significant lower BDNF content compared with HZ group and PHN group(I-J=-38.30,P<0.05;I-J=-33.38,P<0.05),HZ group has no significant difference with PHN group(I-J=4.96,P>0.05).5.Comparisons of BDNF and S100 B in HZ group: The levels of BDNF and S100 B in HZ group were significantly higher than those in normal group,both before treatment and after treatment(I-J = 232.50,27.38,I-J = 164.84,21.46,P <0.05),and the level after treatment was significantly lower than that before treatment(I-J = 205.11,P <0.05;I-J = 143.39,P <0.05).Conclusion: 1.Extraction of DNA by centrifugal column method for q PCR is superior to boiling method.2.Nested PCR production of the standard has a better stability.3.The q PCR method established in this paper is accessible,sensitive and inexpensive,and can be used preferentially.4.The positive detection rate of VZV DNA in PBMC of varicella patients was higher than that in HZ and PHN patients,and based on the fact of the Plasma BDNF,S100 B content is lower than that in HZ,PHN patients,we may conclude that VZV DNA in PBMC of varicella patients is higher,but the nerve injury is milder than that in HZ,PHN patients.5.The VZV DNA positive rate in PBMC and plasma of HZ patients have no significant difference,and the plasma is more convenient for clinical examination. |
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