猪流行性腹泻病毒SYBR Green I实时荧光定量PCR检测方法的建立

为了建立高效、灵敏的猪流行性腹泻病毒(PEDV)检测方法,本研究从GenBank数据库中获取PEDV N基因序列,扩增出PEDV N基因标准质粒,并在N基因的保守区域内设计了一对特异性荧光定量引物,成功建立了SYBR Green I实时荧光定量PCR检测方法。经过一系列试验表明,该检测方法线性关系良好,R^(2)值为0.99;特异性强,敏感性高,最低可检测至2.23 copies/μL,比普通PCR灵敏约100倍;重复性好,组内变异系数为0.25%~0.43%,组间变异系数为0.67%~0.97%;对于各地区96份临床样品检测出PEDV阳性率为25%。本研究建立的实时荧光定量PCR检测方法为PEDV的临床诊断、流行病学调查以及定量研究提供了有效的检测工具。

Infiltration,runoff,and slope stability behaviors of infinite slope with macropores based on an improved Green–Ampt model

Infiltration–runoff–slope instability mechanism of macropore slope under heavy rainfall is unclear.This paper studied its instability mechanism with an improved Green–Ampt(GA)model considering the dual-porosity(i.e.,matrix and macropore)and ponding condition,and proposed the infiltration equations,infiltration–runoff coupled model,and safety factor calculation method.Results show that the infiltration processes of macropore slope can be divided into three stages,and the proposed model is rational by a comparative analysis.The wetting front depth of the traditional unsaturated slope is 17.2%larger than that of the macropore slope in the early rainfall stage and 27%smaller than that of the macropore slope in the late rainfall stage.Then,macropores benefit the slope stability in the early rainfall but not in the latter.Macropore flow does not occur initially but becomes pronounced with increasing rainfall duration.The equal depth of the wetting front in the two domains is regarded as the onset criteria of macropore flow.Parameter analysis shows that macropore flow is delayed by increasing proportion of macropore domain(ω_(f)),whereas promoted by increasing ratio of saturated permeability coefficients between the two domains(μ).The increasing trend of ponding depth is sharp at first and then grows slowly.Finally,when rainfall duration is less than 3 h,ωf andμhave no significant effect on the safety factor,whereas it decreases with increasingωf and increases with increasingμunder longer duration(≥3 h).With the increase ofω_(f),the slope maximum instability time advances by 10.5 h,and with the increase ofμ,the slope maximum instability time delays by 3.1 h.

猪脑心肌炎病毒SYBR GreenⅠ real-time PCR检测方法的建立

针对猪脑心肌炎病毒(EMCV)VP1基因序列设计并合成了1对引物,以构建的含有该引物扩增序列的重组质粒作为阳性标准品,建立了检测EMCV核酸的SYBR GreenⅠreal-time PCR方法。检测结果显示,该方法线性关系好,标准曲线的相关系数达到0.991;对初始模板的检出下限为1×101copies/μL,比常规PCR方法高100倍;与其他猪源病毒,如口蹄疫病毒(FMDV)、猪生殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)均不发生交叉反应;组内与组间的变异系数均小于3%。应用建立的方法检测了17份临床可疑病例的心、脑组织样本,结果1份为阳性。表明,建立的real-time PCR检测方法灵敏度高、特异性强、重复性好,可以用于EMCV的病原检测及定量分析。

猪凋亡细胞因子Fas、FasL、TNFR1和TNF-α SYBR GreenⅠ real-time PCR检测方法的建立

分别针对猪凋亡细胞因子Fas、FasL、TNFR1、TNF-α基因以及看家基因β-actin的序列设计了1对特异性引物,构建含有各自引物扩增序列的重组质粒作为阳性标准品,建立了检测Fas、FasL、TNFR1、TNF-α及β-actin的SYBR GreenⅠreal-ti me PCR方法。该方法线性关系好(r2≥0.995),敏感性高,初始模板的检出下限除TNFR1为1×102copies/μL外,其他均为1×101copies/μL;特异性强,扩增产物形成单一的特异性熔解峰;重复性好,组内与组间的变异系数均小于3%。应用所建立的方法对猪生殖与呼吸综合征病毒感染仔猪外周血单个核细胞中Fas、FasL、TNFR1和TNF-αmRNA的表达水平进行了检测。结果表明,建立的real-ti me PCR检测方法灵敏度高、特异性强、重复性好。

利用SYBR Green检测衣原体Real-Time PCR方法的建立

利用SYBR Green建立了检测种特异性衣原体的Real-Time PCR方法。本方法应用衣原体种特异性的高度保守特异引物，能够扩增627bp特异片段；使用定量标准基因组DNA，本方法能准确检测最少250fg衣原体DNA。Real-TimePCR方法与免疫荧光方法的检测结果表明：检测4种衣原体临床样本，Real-TimePCR敏感性均在96％～98%；特异性均为100％；这两种方法符合率达97％以上（n=60）；批内和批间重复性试验结果表明，本方法具有良好的准确性。本方法的建立对于快速、准确检测临床样本种特异性衣原体提供了一种切实有效的方法。

高致病性PRRSV Nsp2变异株Power SYBR Green real time RT-PCR检测方法的建立

根据高致病性猪生殖与呼吸综合征病毒（PRRSV）Nsp2变异株Nsp2基因保守区的核苷酸序列设计合成了1对引物,建立了Power SYBR Green模式的实时荧光定量RT-PCR方法,用于检测高致病性PRRSV Nsp2变异株。以pMD-Nsp2质粒为阳性对照制作标准曲线,并对该方法的特异性、敏感性、重复性进行评价。结果显示,该方法只能检测到高致病性PRRSV Nsp2变异株的特异性扩增信号;检测线性范围广,在模板浓度为3.15×10^9-3.15×10^0copies/μL具有良好的线性关系,相关系数（r^2）为0.998;最低检测限为3.15copies/μL。对25份疑似高致病性PRRS猪病料的检测结果显示,该方法与病毒分离的符合率为100%。表明,建立的Nsp2-Power SYBR Green RT-PCR方法具有良好的特异性、敏感性和重复性。

微小隐孢子虫和安氏隐孢子虫SYBR Green real time PCR检测方法的建立

根据GenBank中登录的隐孢子虫小亚基rRNA基因序列设计1对引物,并以标准基因组DNA为模板,初步建立了检测乳牛微小隐孢子虫和安氏隐孢子虫的SYBR Green real time PCR方法,并对乳牛微小隐孢子虫和安氏隐孢子虫阳性样品和上海40份乳牛粪便进行了检测。结果表明,此次建立的real timePCR对微小隐孢子虫和安氏隐孢子虫均能扩增出曲线,且其他寄生虫(鸡贝氏隐孢子虫、刚地弓形虫、犬新孢子虫)和大肠杆菌均未检测到;标准基因组DNA的检测阈值达到5个拷贝,牛粪中卵囊的最低检测量为每克粪便5个卵囊,乳牛粪便阳性率为15%(6/40)。表明,建立的荧光定量PCR快速、特异、敏感,可用于乳牛隐孢子虫病的流行病学调查。

猪IL-2、IL-12p40和IFN-γ SYBR Green Ⅰ real-time PCR检测方法的建立

针对猪Th1型细胞因子IL-2、IL-12p40、IFN-γ以及管家基因β-actin的基因序列分别设计一对特异性引物,构建含有各自引物扩增序列的重组质粒作为阳性标准品,建立了检测IL-2、IL-12p40、IFN-γ及β-actin的SYBR Green Ⅰ real-ti mePCR方法。该方法线性关系好,标准曲线的相关系数均达到0.997以上;敏感性高,初始模板的检出下限均达到100拷贝/μL;特异性强,扩增产物形成单一的特异性熔解峰;重复性好,组内与组间的变异系数均小于3.5%。应用所建立的方法对猪繁殖与呼吸综合征病毒感染仔猪外周血单个核细胞中IL-2、IL-12p40和IFN-γ mRNA的表达水平进行了检测。结果表明,所建立的real-time PCR检测方法灵敏度高、特异性强、重复性好。

Monochromatic green light stimulation during incubation shortened the hatching time via pineal function in White Leghorn eggs

Background:Effect of monochromatic green light illumination on embryo development has been reported in chickens.The avian pineal gland is an important photo-endocrine organ formed by a mediodorsal protrusion during embryonic development.However,the involvement of pineal gland in the light transduction process remains to be elucidated.In the present study,we investigated the influence of monochromatic green light on hatching time and explored the possible mechanism via pineal function.Results:A total of 600 eggs of White Leghorn(Shaver strain)were incubated under photoperiods of either 12 h of light and 12 h of darkness using monochromatic green light(12L:12D group)or 24 h of darkness(0L:24D group)for 18 d.Compared to 0L:24D group,the green light stimulation shortened the hatching time without extending the hatch window or impairing hatchability.The liver of embryos incubated in the 12L:12D light condition was heavier than those of the 0L:24D group on d 21 post incubation which may be linked to the observed increase in the serum concentration of insulin-like growth factor 1(IGF-1);primary secretion of the liver.Histological structure analysis of pineal gland demonstrated that the light stimulation increased follicle area,wall thickness and lumen area on d 10 and d 12 post incubation.Rhythmic function analysis demonstrated that three clock related genes(brain and muscle ARNT-like-1,BMAL1;circadian locomotor output cycles kaput,CLOCK;and cryptochrome-1,CRY1)and a melatonin rate-limiting enzyme related gene(arylalkylamine N-acetyltransferase,AANAT)were rhythmically expressed in the pineal gland of the 12L:12D group,but not in the 0L:24D group.Simultaneously,the light stimulation also increased the concentration of melatonin(MT),which was linked to hepatocyte proliferation and IGF-1 secretion in previous studies.Conclusions:The 12L:12D monochromatic green light stimulation during incubation shortened hatching time without impairing hatching performance.Pineal gland’s early histological development and maturation of its rhythmic function were accelerated by the light stimulation.It may be the key organ in the photo-endocrine axis that regulates embryo development,and the potential mechanism could be through enhanced secretion of MT in the 12L:12D group which promotes the secretion of IGF-1.

SYBR Green Ⅰ Real-time PCR检测IFN-CSP对HepG2.2.15细胞内HBV-DNA影响

目的建立SYBR GreenⅠReal-time PCR检测HBV-DNA方法,并检测IFN-CSP对HepG2.2.15细胞内HBV-DNA影响。方法提取HepG2.2.15细胞DNA,PCR扩增后纯化,PCR纯化产物梯度稀释作为标准品,采用SYBR GreenⅠReal-time PCR检测,建立标准曲线,并分析方法的特异性、灵敏度、重复性和稳定性。运用建立的SYBR GreenⅠ方法检测IFN-CSP对HepG2.2.15细胞内HBV-DNA影响,并与Taqman方法进行比较。结果 SYBR GreenⅠReal-time PCR方法特异性、重复性及稳定性均较好,线性范围为107~102copies,检测灵敏度可达102copies。IFN-CSP对HepG2.2.15细胞内HBV-DNA具有抑制效果,且呈剂量依赖性。SYBR GreenⅠ方法与Taqman商业试剂盒检测结果差异无统计学意义。结论 SYBR GreenⅠReal-time PCR方法简便快速、结果准确、价格低廉,可以满足HBV-DNA拷贝数检测的需要。

Source time functions of the Gonghe，China earthquake retrieved from long－period digital waveform data using empirical Green＇s function technique

An earthquake of Ms= 6, 9 occurred at the Gonghe, Qinghai Province, China on April 26, 1990. Three larger aftershocks took place at the same region, Ms= 5. 0 on May 7, 1990, Ms= 6. 0 on Jan. 3, 1994 and Ms= 5. 7on Feb. 16, 1994. The long-period recordings of the main shock from China Digital Seismograph Network (CDSN) are deconvolved for the source time functions by the correspondent0 recordings of the three aftershocks asempirical Green's functions (EGFs). No matter which aftershock is taken as EGF, the relative source time functions (RSTFs) Obtained are nearly identical. The RSTFs suggest the Ms= 6. 9 event consists of at least two subevents with approximately equal size whose occurrence times are about 30 s apart, the first one has a duration of 12 s and a rise time of about 5 s, and the second one has a duration of 17 s and a rise time of about & s. COmParing the RSTFs obtained from P- and SH-phases respectively, we notice that those from SH-phases are a slightly more complex than those from p-phases, implying other finer subevents exist during the process of the main shock. It is interesting that the results from the EGF deconvolution of long-Period way form data are in good agreement with the results from the moment tensor inversion and from the EGF deconvolution of broadband waveform data. Additionally, the two larger aftershocks are deconvolved for their RSTFs. The deconvolution results show that the processes of the Ms= 6. 0 event on Jan. 3, 1994 and the Ms= 5. 7 event on Feb. 16,1994 are quite simple, both RSTFs are single impulses.The RSTFs of the Ms= 6. 9 main shock obtained from different stations are noticed to be azimuthally dependent, whose shapes are a slightly different with different stations. However, the RSTFs of the two smaller aftershocks are not azimuthally dependent. The integrations of RSTFs over the processes are quite close to each other, i. e., the scalar seismic moments estimated from different stations are in good agreement. Finally the scalar seismic moments of the three aftershocks are compared. The relative scalar seismic moment Of the three aftershocks deduced from the relative scalar seismic moments of the Ms=6. 9 main shock are very close to those inverted directly from the EGF deconvolution. The relative scalar seismic moment of the Ms =6. 9 main shock calculated using the three aftershocks as EGF are 22 (the Ms= 6. 0 aftershock being EGF), 26 (the Ms= 5. 7 aftershock being EGF) and 66 (the Ms= 5. 5 aftershock being EGF), respectively. Deducingfrom those results, the relative scalar sesimic moments of the Ms= 6. 0 to the Ms= 5. 7 events, the Ms= 6. 0 tothe Ms= 5. 5 events and the Ms= 5. 7 to the Ms= 5. 5 events are 1. 18, 3. 00 and 2. 54, respectively. The correspondent relative scalar seismic moments calculated directly from the waveform recordings are 1. 15, 3. 43, and 3. 05.

Fast Evaluation of Time-Domain Green Function for Finite Water Depth

For computation of large amplitude motions of ships fastened to a dock, a fast evaluation scheme is implemented for computation of the time-domain Green function for finite water depth. Based on accurate evaluation of the Green function directly, a fast approximation method for the Green function is developed by use of Chebyshev polynomials. Examinations are carried out of the accuracy of the Green function and its derivatives from the scheme. It is shown that when an appropriate number of polynomial terms are used, very accurate approximation can be obtained.

SYBR Green I染料real-time RT-PCR检测Rb Ap46基因表达条件的优化

目的 通过优化反应体系,建立SYBRGreenI染料real timeRT PCR检测RbAp4 6基因表达的方法。方法 构建pUCm Tvector RbAp4 6阳性模板,根据分子量及阿佛加德罗常数(6 .0 2 3×10 2 3 分子数/mol)换算为分子拷贝数,10倍倍比稀释后做标准曲线。根据标准曲线的斜率、相关系数、荧光强度及熔解曲线优化反应体系中各组分的量及反应条件。结果 2 5 μl的反应体系中10×PCR缓冲液2 .5 μl,2 5mmol/LMgCl2 2 .2 μl,10mmol/LdNTPs0 .5 μl,2 0×SYBRGreenI 0 .5 μl,12 .5umol/L引物各0 .5 μl,5U/μlTaqDNA聚合酶0 .3μl,cDNA1μl(相当于5 0ngRNA) ,双蒸水17μl。反应条件为:94℃5min预变性,96℃2 0s变性,5 8℃30s退火,72℃4 5s延伸,4 0个循环,80℃时采集荧光信号,可以获得最好的扩增效率。结论 优化后的SYBRGreenIreal timePCR适合于检测RbAp4 6基因表达,具有比较好的重复性,灵敏度高,特异性强。

奶牛隐孢子虫SYBR Green Real-time PCR检测方法的建立

为建立一种方便快捷的检测奶牛隐孢子虫的实时荧光定量PCR(Real-time PCR)方法,根据GenBank中已发表的隐孢子虫18s rRNA基因设计一对引物,并以从卵囊中提取的DNA为模版,初步建立了检测奶牛隐孢子虫的SYBR Green Real-time PCR方法,进而对采集的奶牛粪便进行了特异性、敏感性和重复性检测。结果显示,本研究建立的Real-time PCR方法敏感性高,最低检测阈为10个拷贝的质粒DNA和1g粪便中的10个卵囊,扩增产物的特异性良好,重复性试验的标准差为0.494,重复性良好。研究表明,建立的Real-time PCR快速、特异、敏感,可用于奶牛隐孢子虫病的流行病学调查。

Effects of sowing time on bolting and returning green in welsh onion

Qiyechangbai and Hadacongl were used to study the relationship of the leaf age of overwinter to bolting and, returning green by the difference of sowing time. The results showed that the earlier the seeds were sown, the older leaf age of overwinter was, the higher the rates of returning green and bolting rate were, the earlier the bolting time was. The leaf age of overwinter of Qiyechangbai sown August 31 to September 14 was 2.1-3.1 leaves and the rate of returning green was 86.5%-92.1%, while the leaf age of overwinter of Hadacongl sown September 7 to September 14 was 2.3-2.7 leaves and the rate of returning green was 88.5%-93.8%, both varieties didn＇t bolt. In addition, in the same sowing time, the bolting rate of Hadacong I was higher than that of Qiyechangbai slightly.

Rapid genotyping of human rotavirus using SYBR green real-time reverse transcription-polymerase chain reaction with melting curve analysis

AIM: To develop a real-time reverse transcriptionpolymerase chain reaction(RT-PCR) assay to genotype rotavirus(G and P) in Alberta from January 2012 to June 2013. METHODS: We developed and validated a different approach to perform rotavirus G and P genotyping using a two-step SYBR green RT-PCR(rt-g PCR) by selecting genotype-specific primers of published conventional RT nested PCR(cn RT-PCR) assay and optimizing the amplification conditions. c DNA was first synthesized from total RNA with Super Script? Ⅱ reverse transcriptase kit followed by amplication step using monoplex SYBR green real-time PCR. After the PCR reaction, melting curve analysis was used to determine specific genotype. Sixteen samples previously genotyped using cn RT-PCR were tested using the new assay and the genotyping results were compared as sensitivity analysis. Assay specificity was evaluated by testing other gastroenteritis viruses with the new assay. The amplicon size of each available genotype was determined by gelelectrophoresis and DNA sequences were obtained using Sanger-sequencing method. After validation and optimization, the new assay was used to genotype 122 pediatric clinical stool samples previously tested positive for rotavirus using electron microscopy between January2012 and June 2013.RESULTS: The new rt-g PCR assay was validated and optimized. The assay detected G1 to G4, G9, G12 and P[4] and P[8] that were available as positive controls in our laboratory. A single and clear peak of melting curve was generated for each of specific G and P genotypes with a Tm ranging from 80 ℃ to 82 ℃. The sensitivity of rt-g PCR was comparable to cn RT-PCR with 100% correlation of the 16 samples with known G and P genotypes. No cross reaction was found with other gastroenteritis viruses. Using the new rt-g PCR assay, genotypes were obtained for 121 of the 122 pediatric clinical samples tested positive for rotavirus: G1P[8](42.6%), G2P[4](4.9%), G3P[8](10.7%), G9P[8](10.7%), G9P[4](6.6%), G12P[8](23.0%), and unknown GP[8](0.8%). For the first time, G12 rotavirus strains were found in Alberta and G12 was the second most common genotype during the study period. Gel electrophoresis of all the genotypes showed expected amplicon size for each genotype. The sequence data of the two G12 samples along with other genotypes were blasted in NCBI BLAST or analyzed with Rota C Genotyping tool(http://rotac.regatools.be/). All genotyping results were confirmed to be correct.CONCLUSION: rt-g PCR is a useful tool for the genotyping and characterization of rotavirus. Monitoring of rotavirus genotypes is important for the identification of emerging strains and ongoing evaluation of rotavirus vaccination programs.

猪圆环病毒1型、2型、3型SYBR GreenⅠ实时荧光定量PCR鉴别方法的建立及应用

猪圆环病毒(PCV)包括猪圆环病毒1型(PCV1)、猪圆环病毒2型(PCV2)、猪圆环病毒3型(PCV3)。近些年来,PCV1、PCV2、PCV3基因型之间存在共感染,因此建立一种快捷、特异、敏感的PCV1、PCV2、PCV3的SYBR GreenΙ实时荧光定量PCR鉴别检测方法显得尤为必要。本试验通过特异性引物筛选,各项反应条件优化,建立了实时荧光定量PCR鉴别方法。结果表明:PCV1、PCV2、PCV3检测下限分别为40.3、25.2、22.4 copies/μL。与猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)均无交叉反应。批内及批间变异系数均小于1%。对98份PCV疑似阳性病料检测结果表明,PCV1、PCV2、PCV3感染率分别为10.2%、65.3%、53.1%,共感染率为7.1%。该方法具有良好的特异性、敏感性和稳定性,为PCV1、PCV2、PCV3的早期检测、定量检测及后期研究提供了技术手段。

Green Function of Generalized Time Fractional Diffusion Equation Using Addition Formula of Mittag-Leffler Function

In this paper, we use the Mittag-Leffler addition formula to solve the Green function of generalized time fractional diffusion equation in the whole plane and prove the convergence of the Green function.

SYBR Green I Realtime PCR检测胃癌组织中LIN28基因方法的建立

目的建立SYBRGreen I荧光染料实时定量PCR方法，测定LIN28基因在胃癌组织中的表达水平，并进行实验室方法学评价。方法采用RT—PCR扩增LIN28基因片段，将该片段克隆到质粒载体PMD18-T上，转化入大肠埃希氏菌（DH5a）中，PCR鉴定后，提取重组质粒，以阳性质粒为模板，建立SYBR—GreenI荧光定量PCR标准曲线，以pactin为内参，运用Rotor—Gene6000seriessoftware做相对定量分析，检测30例胃癌患者的癌组织中LIN28的表达水平。结果用重组质粒制作的标准曲线循环阂值与模板浓度有良好的线性关系，内参pactin和LIN28标准曲线相关系数分别为0．9997和0．9991，其最低的检测拷贝数为1，LIN28溶解曲线呈单个特异峰。结论成功地构建了LIN28的原核表达载体。建立的SYBRGreenI荧光染料实时定量PCR法特异度、敏感度高，稳定性好，可用于定量测定胃癌组织中LIN28的表达水平。

A New Formula to Obtain Exact Green＇s Functions of Time-Dependent SchrōdingerEquation

We obtain a new relation between Green's functions of the time-dependent Schrōdinger equation forstationary potentials and Green's functions of the same equation for certain time-dependent potentials. The relationobtained here emerges very easily from a transformation introduced by Ray [J.R. Ray, Phys. Rev. A26 (1982) 729] andgeneralizes former work of Dodonov et al. [V.V. Dodonov, V.I. Man'ko, and D.E. Nikonov, Phys. Lett. A162 (1992)359.]