3.Study on the amplifying and typing of low copy number DNA
Chinese Journal of Forensic Medicine 2009;24(6):383-385,403
Objective To study the influence on STR typing for low copy number(LCN)DNA using different methods of amplifcation and detection.Methods Control DNA 9947A was diluted and then amplifled with Identifiler~(TM) or DNATyper15~(TM).The heat cycles were set to 28 or 28 add 6.Each template wag amplified three times in parallel,and then the amplified products or the product mixture of three amplifictions were deteced with 310 or 3130 Genetic Analyzer.Results The success rate of STR typing with the method 28+6 cycles was higher than that of 28 cycles.There are no correlation between the allele imbalance or allele dropout and STR locus.With the reduction in the amount of template DNA,allele imbalance and dropout gradually increased,and the allele dropout was more common than allele imbalance when the amount of template DNA Wag very small.When the product mixture of three amplifictions were deteced.the occurrence of allele imbalance and dropout reduce obviously.Conclusion The success rate of STR typing of LCN DNA can be obviously increased by detecting the product mixture of three amplifictions in parallel combined with the 28+6 heat cycle condition.
4.Preparation of internal quality control substance of real-time PCR to analyze UU-DNA and the design quality control programs
Yanmei GE ; Di ZHANG ; Suyi FAN ; Hang YUAN ; Yuan MAO
International Journal of Laboratory Medicine 2016;37(8):1070-1071,1074
Objective Developing an internal quality control substance of Ureaplasma urealyticum(UU)‐DNA for real‐time PCR to establish an internal quality control system and preliminary evaluation its clinical value .Methods Internal quality control sub‐stance was prepared by mixing samples which Ct value were 24-25(positive sample) and 32 -33(weak positive sample) ,respec‐tively .At the same time ,selecting samples that test results were negative as negative control .The target value ,standard deviation (s) and coefficient of variation(CV) of internal quality control substance were defined by“instant method”for the first 20 runs and Levey‐Jennings quality control(QC) chart after the first 20 runs .Using the“Westdard” multi‐rule quality control methods to ana‐lyze the detection results .Exporting OPSPecs chart by quality control rules in Unity Real Time (URT ) system and setting up new quality control rules according with OPSPecs chart .Results 131 times of the detection of quality control substance were performed totally .The first 20 runs were defined by“instant method”and later 111 runs were defined by Levey‐Jennings QC chart ,the results were stable of quality control substance and reasonable quality control rules .Conclusion Preparing of internal quality control sub‐stance of UU‐DNA used in real‐time PCR might be easy and stable .So ,the internal quality control substance of UU‐DNA could be worthy for practical application in this PCR laboratory .Design internal quality control rules based OPSPecs chart in molecular de‐tection is very simple and practical .
5.HMGB1 upregulates P-glycoprotein expression in the brain microvascular endothelial cells of the mouse
Yan CHEN ; Nian YU ; Yuan XIE ; Kang ZHANG ; Qing DI
Journal of Medical Postgraduates 2015;(2):141-145
[Abstract ] Objective High-mobility group box-1 (HMGB1) is abundantly released in the epileptogenic brain tissue , but few reports are seen about the effect of HMGB 1 on the expression of P-glycoprotein ( P-gp) in the vascular endothelial cells of the epi-leptogenic tissue .This study is to explore whether HMGB 1 can regulate P-gp expression in the brain microvascular endothelial cells of the mouse in vitro . Methods Immortalized brain microvascular endothelial bEnd .3 cells of the mouse were cultured in vitro and al-located to different concentration groups ( treated with culture medium containing 10 , 100 , 500 , and 1000 ng/mL HMGB1 for 8 hours), treatment duration groups (treated with culture medium containing 100 ng/mL HMGB1 for 4, 8, 16, 24, and 32 hours), and a control group ( treated with culture medium without HMGB 1 ) .The mRNA expression of P-gp-encoding gene-multidrug resistance gene 1a (mdr1a) was detected by real-time qPCR, and its protein expression determined by Western blot and immunocytochemistry . Results The results of qPCR manifested that the expressions of mdr 1a mRNA were 1.646 ±0.176, 1.777 ±0.135, 1.617 ±0.043, and 1.398 ±0.182 in the 10, 100, 500, and 1000 ng/mL HMGB1 groups, respectively, significantly higher than 1.030 ±0.284 in the control group (P<0.05), and so were those in the 4, 8, 16, 24 h, and 32 h groups (2.655 ±0.112, 2.168 ±0.212, 1.823 ± 0.232, 1.418 ±0.376, and 1.445 ±0.123) than in the control (1.010 ±0.164) (P <0.05).Western blot showed a significant increase in the P-gp protein expression in all the concentration groups (P<0.05) as well as in the 8 h and 16 h treatment duration groups as compared with the control group (P<0.05).Immunocytochemis-try also revealed a higher P-gp expression in the HMGB1-treated than in the control cells (P<0.01). Conclusion HMGB1 can upregu-late the expressions of mdr1a mRNA and P-gp protein in the brain microvascular endothelial cells of the mouse , which may associated with drug resistance of central nervous system diseases , especially that of epilepsy .
6.Inhibitory effects of andrographis paniculata and silybum marianum on the efflux pump of MRSA 41577
Linhui YUAN ; Di WANG ; Kunpeng XIE ; Mingjie XIE
Chinese Journal of Biochemical Pharmaceutics 2016;36(11):27-29
Objective To study the inhibitory effects of andrographis paniculata and silybum marianum on the efflux system of MRSA 41577. Methods Inhibitory effects of andrographis paniculata and silybum marianum on efflux system of MRSA 41577 was evaluated using fluorescence spectrophotometry.PCR was applied to detect the norA efflux gene.By RT-PCR method for detection of andrographis paniculata and silybum marianum influence of the expression of norA efflux gene.Results Andrographis paniculata and silybum marianum significantly increased the accumulation of ciprofloxacin in MRSA 41577 in a time-dependent manner.At 12 minute, andrographis paniculata and silybum marianum respectively increased ciprofloxacin in MRSA41577 by 49% and 76%( P <0.05 ) , which is superior to that of reserpine. Further mechanism studies indicated that andrographis paniculata and silybum marianumcould reduce the expression of norA in MRSA 41577.After incubated with andrographis paniculata and silybum marianum for 16 h, the relative expression of norA of MRSA41577 was respectively reduced by 35% and 42% ( P <0.05 ). Conclusion Andrographis paniculata and silybum marianumcould inhibit MRSA efflux system through reducing pathogen ’s expression of norA and NorA protein.
8.Analysis of clinical characteristics and JAK2V617F mutation of Tibetan people living at high altitudes with polycythemia.
Di-jiao TANG ; Yuan-xin YE ; Jun LI
Chinese Journal of Hematology 2012;33(11):960-962
Adult
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Altitude
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Asian Continental Ancestry Group
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genetics
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Female
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Humans
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Janus Kinase 2
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genetics
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Male
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Middle Aged
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Mutation
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Polycythemia
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diagnosis
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ethnology
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genetics
9.Influence of subculture cycle on tube plantlet growth of Dendrobium huoshanense and medium composition
Shuhua JIA ; Di WANG ; Yuan GAO ; Yongping CAI ; Yi LIN
Chinese Traditional and Herbal Drugs 1994;0(08):-
Objective To optimize the best subculture cycle through studying the influence of subculture time on tube plantlet growth of Dendrobium huoshanense and the change of medium composition.Methods Height,tiller,fresh weight,and chlorophyll content of the tube plantlet and pH value,water content,and sugar content of the medium were measured after different cycles of the subculture,the cost of culture medium for subculture was calculated as well.Results The height of the tube plantlet increase 282.86%,the tiller increase by 3.5 times,fresh weight reaches its maximum,chlorophyll content of the tube plantlet almost reaches its maximum after 40 d subculture;while water content and sugar content of the medium are decreased to the lowest point,pH value of medium(
10.Association of HLA-DR/DQ with Oral Lichen Planus
hui-yuan, LI ; ling-di, XU ; guo-yao, TANG
Journal of Shanghai Jiaotong University(Medical Science) 2006;0(10):-
Objective To investigate the association of human leukocytes antigen(HLA)-DR/DQ with oral lichen planus(OLP) in the area of Yangtze River Delta. Methods HLA-DRB1 and DQB1 genotyping of 44 unrelated OLP patients and 150 normal controls were performed by polymerase chain reaction-sequence specific primers(PCR-SSP) method.The data were compared between the OLP group and normal controls,and between different types of OLP patients. Results The frequency of HLA-DRB1*09 and HLA-DRB1*07 alleles were significantly higher in OLP group than those in normal controls(56.8% vs 31.3% and 27.3% vs 13.3%,P