CHIKV Real-time PCR检测方法的建立及试剂盒研发

目的建立快速特异检测基孔肯雅病毒的Real-time PCR方法及试剂盒。方法根据发表在Genbank上15株基孔肯雅病毒基因序列全长,应用ClustalW2.0和DNAMAN8软件筛选出位于其E1基因上的种内保守种间特异的目的片段,并据此设计出最优引物。人工合成该基因片段并构建重组质粒,对重组质粒梯度稀释后,再利用SYBR Green I Real-time PCR的方法检测该特异基因的浓度,建立标准品曲线,用于临床上基孔肯雅病毒的早期诊断。结果琼脂糖凝胶电泳结果显示,标准品的PCR扩增产物的电泳条带长度与目标条带一致,测序结果显示与目标条带序列一致,说明引物与阳性质粒性能良好。经优化确定CHIKV荧光定量PCR反应体系中最佳的引物浓度为350 nmol/L,最佳的反应条件为：50 ℃ 2 min;95 ℃预变性2 min;以95 ℃ 15 s,60 ℃ 15 s,72 ℃ 1 min进行40个循环扩增,在72 ℃进行荧光采集。根据荧光定量PCR熔解曲线出现特异性单峰,并对黄病毒属成员登革病毒和寨卡病毒的检测为阴性,表明该检测方法特异性高;检测阈值达302 拷贝 ,显示敏感性好,重复试验的变异系数均小于0.1%,说明该实验设计稳定性良好。结论基于该方法的试剂盒具有特异性好、灵敏度高、重复性好、能够快速定量等优点,有利于临床上基孔肯雅病毒的早期诊断,具有良好的应用前景。     

实时荧光定量PCR检测外周血TREC的方法研究

目的建立及优化检测移植后患者外周血TREC拷贝数的实时荧光定量PCR（FQ-PCR）的方法及实验影响条件，为临床研究造血干细胞移植的免疫功能重建机制提供方法学基础。方法在ABI7000 PCR仪上实时扩增检测构建的TREC质粒标准品，摸索最佳扩增条件及建立标准曲线，然后用优化的FQ-PCR方法检测临床样本。结果T3、T4引物和R3、R4引物的扩增效率分别优于T1、T2引物和R1、R2引物。TaqMan-MGB探针比普通的TaqMan探针扩增效率高。金牌热启动Taq酶，比较普通Taq酶而言。提高了PCR的特异性和敏感性。FQ-PCR的反应条件设定：95℃10min后95℃5s，53℃30s，40循环结束。结论通过摸索与优化实验条件，建立了实时荧光定量PCR检测外周血单个核细胞中TREC的方法。     

小鼠诺如病毒荧光定量PCR检测方法的建立及初步应用

目的建立小鼠诺如病毒(murine norovirus,MNV)实时荧光定量PCR检测方法,为建立MNV规范化检测方法提供依据。方法根据NCBI公布的60个MNV病毒株核酸序列设计特异性引物,构建标准阳性质控品,建立MNV实时荧光定量PCR方法,并进行特异性、灵敏度、重复性及稳定性评价。用该方法对766只送检小鼠的盲肠内容物样品进行检测,初步了解北京地区实验小鼠MNV感染状况。结果成功建立MNV实时荧光定量PCR检测方法,该方法特异性好,与同种属人类诺如病毒(Hu No Vs)、猫杯状病毒(FCV)均无交叉反应,检测灵敏度可达101copies/μL。批内及批间CT值变异系数均小于2%。应用该方法在766份小鼠盲肠内容物样品中检出301份MNV核酸阳性样品,阳性率39.3%。结论建立的MNV实时荧光定量PCR检测方法特异性好,灵敏度高,重复性好,可以用于MNV的快速定量检测。     

一种针对蓝舌病毒两个不同基因片段的荧光定量PCR检测技术

目的:建立一种高灵敏度、高特异性的蓝舌病毒RNA荧光定量RT-PCR检测技术,能够检测不同血清型蓝舌病毒(BTV)。方法:应用改良的BTV双链RNA变性方法,针对BTV基因组片段1和5的相对保守序列设计2对特异性引物,建立了BTV荧光定量RT-PCR检测方法,并对该方法进行灵敏度、特异性、重复性评价。结果:该方法能检测11种不同血清型BTV,其灵敏度可达到10copies/μl;使用所设计引物的荧光定量PCR能特异地扩增BTV,其Ct值差异较小,并与阴性对照EHDV-5无交叉反应;重复检测CV值小于5%。结论:检测方法具有灵敏度高、特异性强等优点,适用于BTV的快速检测和诊断。     

高致病性禽流感通用和H5亚型荧光定量PCR方法的建立

目的:建立高致病性禽流感H5亚型荧光定量PCR方法.方法:根据禽流感病毒(AIV)M基因和H5-HA基因分别设计AIV通用和H5亚型特异荧光定量PCR引物和探针,优化反应体系和反应条件,分析该试剂的敏感性、特异性、重复性等.结果:经优化反应体系和反应条件后,AIV通用试剂和AIV-H5试剂的敏感性达到1×101.25EID50水平;与细胞培养比较,AIV-H5试剂Kappa吻合系数κ为0.991(P<0.01),统计学显示两种检测结果的吻合度很强;批内和批间精密度CT值的CV值均远小于10%,可重复性良好.结论:本项目研制的AIV通用试剂和AIV-H5试剂的敏感性、特异性、重复性均好,适合在禽流感疫情和疑似H5禽流感病例中检测使用.     

白色念珠菌ITS2的实时荧光定量PCR快速检测方法的建立及评价

目的 建立一种快速鉴定白色念珠菌的实时荧光定量PCR检测方法。方法 在白色念珠菌核糖体RNA编码基因（rDNA）中的内转录间隔区2（ITS2）上设计特异性引物和TaqMan-MGB探针,通过实时荧光定量PCR分析系统建立白色念珠菌的检测方法,对其敏感度、重复性和特异性进行方法学评价,并在模拟白色念珠菌血症的血标本中初步探讨其应用价值。结果 所建方法对白色念珠菌纯菌液的准确检测下限为10¹CFU/ml,相应的批内、批间变异系数（CV）分别为1.57%和2.20%。对135株白色念珠菌临床分离株、其他非白念菌株、细菌临床分离株和血液标本进行检测,结果显示该方法特异性为100%。另外,对于模拟白色念珠菌血症的血标本,该方法的最低检测下限为10²CFU/ml。结论 所建方法具有特异性和敏感度高、重复性好的特性,可适用于临床上白色念珠菌引起的血流感染的快速鉴定。     

建立禽流感H7和H9亚型荧光定量PCR检测方法

目的建立高致病性禽流感H7和H9亚型荧光定量PCR方法。方法根据禽流感病毒(avian influenzavirus,AIV)HA-H7基因和HA-H9基因分别设计特异AIV H7和H9亚型特异荧光定量PCR引物和探针,优化反应体系和反应条件,分析该试剂的敏感性、特异性、重复性等。结果经优化反应体系和反应条件后,AIV H7试剂和AIV H9试剂的敏感性达到1×104.67EID50和1×103.80EID50水平,特异性与细胞培养敏感性类似;批内和批间精密度Ct值的CV值均远小于10%,可重复性良好。结论本项目研制的AIV H7和H9亚型的敏感性、特异性、重复性均好,适合在禽流感H7和H9亚型疫情和人类疑似禽流感病例中检测使用。     

实时荧光定量PCR检测尿液CK20方法的建立

目的:建立一种简便、快速、灵敏度和特异性较好的定量检测尿液CK20方法,用于膀胱移行细胞癌(Transitional cell ear-cinoma of the bladder,TCCB)早期诊断、预后及复发的评估.方法:培养膀胱癌T24细胞,针对CK20基因保守序列设计合成两对引物和Taqman探针,将PCR扩增产物片段克隆,作为定量检测的标准品,优化反应条件,建立快速检测CK20mRNA的实时荧光定量PCR方法,并进行方法学评价.结果:荧光定量PCR检测尿液CK20方法能较好的定量枪出尿液CK20mRNA,检测范围为102～102copies/μ1,灵敏度为102 copies/μ1,特异性好,批内、天间重复性(Coefficient of variation,CV)分别为0.89%和2.45%.结论:成功建立了快速检测尿液CK20的荧光定量PCR方法,为进一步开发出用于TCCB早期诊断、预后及复发评估的定量检测试剂盒奠定了基础.     

柯萨奇B组3型病毒SYBR Green Ⅰ RT-PCR 检测方法的建立

目的 建立一种灵敏、特异的柯萨奇B组3型(CV-B3)病原SYBR Green Ⅰ RT-PCR实时荧光定量检测方法,用于快速、准确地检测CV-B3。方法 设计柯萨奇B组病毒通用引物以及VP1基因的T7启动子特异性引物,以体外转录获得的RNA为标准品,绘制标准曲线,建立CV-B3的荧光定量PCR检测方法;对检测方法的敏感度、特异性、重复性进行评价。结果该检测方法在10～10⁹ 拷贝/微升范围内具有良好的扩增效率和线性关系,最低检测限为10拷贝/微升,标准曲线r2=0.999, 扩增效率为95.7%;且对柯萨奇病毒A组6型(CV-A6)、柯萨奇病毒A组10型(CV-A10)、肠道病毒71型(EV-71)均无交叉反应,重复性实验变异系数(CV%)<2%。结论 本实验建立的检测方法敏感度较高,同时具备良好的特异性及重复性,可用于CV-B3病原的临床及实验室样本的定性定量检测。     

实时荧光定量TaqMan-PCR检测小鼠多瘤病毒方法的建立

目的 建立一种快速、特异、敏感的小鼠多瘤病毒 (murine polyomavirus, MPyV)荧光定量 TaqMan PCR检测方法, 用于MPyV核酸的检测。 方法 根据GenBank中小鼠多瘤病毒基因组序列设计了一对特异性引物及TaqMan探针, 扩增长度为69 bp的片段, 通过优化反应体系和反应条件, 对构建的重组质粒标准品进行检测, 并绘制标准曲线, 进行特异性、敏感性和重复性试验。最后用建立的方法对人工感染MPyV的肺脏、脾脏和粪便, 及86 份小鼠临床样本进行检测, 验证在临床应用中的效果。 结果 所建立的检测方法特异性强, 只能在小鼠多瘤病毒DNA中检出荧光信号, 检测灵敏度达到100拷贝, 批内和批间重复性好, 检测结果变异系数(CV)均小于1.13%, 人工感染MPyV小鼠的肺脏、脾脏和粪便均为阳性, 对86份临床样本进行检测, 有3份样本呈阳性(阳性率为3.5%)。 结论 建立的MPyV荧光定量TaqMan-PCR检测方法特异性强、敏感性高、重复性好, 适合用于MPyV临床诊断、日常监测和流行病学调查。     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.     

Scientific principles and rigorous processes should be followed in developing clinical guidelines for therapeutic interventions of integrative medicine

A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care（~＇4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.