利用反向PCR方法扩增细菌热激蛋白HSP60基因

利用PCR简并引物扩增出HSP6 0基因中一段约 6 0 0bp的核心片段 ,将该核心片段标记为探针 ,与基因组DNA进行Southern杂交 ,选择出适宜的限制性内切酶 ,以便消化基因组DNA得到大小合适的、含有HSP6 0基因的酶切片段。将酶切片段自身环化后作为模板进行反向PCR ,引物的延伸方向自核心片段出发延环化分子向未知序列区进行 ,可扩增出核心区上下游的序列。应用该方法 ,扩增并测定了寓齿双歧杆菌 (Bifidobacteriumdenticolens)DSM1 0 1 0 5 T、奇异双歧杆菌 (Bifidobacteriuminopinatum)DSM1 0 1 0 7T 和阴道加德纳氏菌 (Gard nerellavaginalis)ATCC1 40 1 8T 的HSP6 0全基因序列及青春双歧杆菌 (Bifidobacteriumadolescentis)JCM1 2 75 T98%以上的HSP6 0全基因序列。结果表明 ,反向PCR方法可有效的扩增细菌HSP6 0基因     

极严重少弱精症患者的新鲜周期移植中卵裂球数目对妊娠结局的影响

目的:探讨极严重少弱精症患者的新鲜周期移植中卵裂球数目对妊娠结局的影响。方法:回顾性分析392个新鲜胚胎移植周期,这些胚胎根据移植天数分为第二天移植(Day2)组68周期和第三天移植(Day3)组324周期,其中Day2组根据卵裂球数分为3个(A组,3周期),3～5个(B组,57周期),5个(C组,8周期),Day3组根据卵裂球数分为7个(D组,33周期),7～9个(E组,242周期),9个(F组,49周期)。每个组又根据胚胎评级分为三个亚组,亚组1均为移植1～2级的胚胎(A1～F1),亚组2均为移植3～4级的胚胎(A2～F2),亚组3为一个1～2级和一个3～4级移植胚胎(A3～F3)。综合比较各组和相应亚组之间的种植率,临床妊娠率,流产率及活产率的差别。结果:①在day2组中,ABC三组的胚胎种植率,临床妊娠率,流产率及活产率均无统计学差异(P0.05)。在Day3组中,E组的胚胎种植率,临床妊娠率及活产率均高于D组,差异具有统计学意义(P0.05)。而D组和F组及E组和F组之间差异均无统计学意义。②在相同卵裂球数组中,不同的胚胎分级各亚组之间(如B1,B2,B3间)的胚胎种植率,临床妊娠率,流产率及活产率均无统计学差异。不同卵裂球数其相应的同一亚组之间(A1,B1,C1等)上述妊娠结局指标无统计学差异。结论:严重少弱精形成的胚胎优先推荐第二天卵裂球数为3～5个及第三天卵裂球数为7～9个的优质胚胎进行移植。     

甘薯丛枝病植原体的PCR检测

以报道的植原体（Phytoplasma)16SrDNA基因保守序列为依据,设计合成了两对引物对R16mF2/R16mR2和R16F2／R16R2,以甘薯丛枝病（SPWB）带病植株的叶脉中提取的DNA为模板,应用聚合酶链式反应（PCR）技术和巢式PCR（Nested-PCR)技术对甘薯丛枝病病原进行分子检测。结果表明PCR扩增出了1．5kb的特异片段,在PCB基础上的巢式PCR扩增出了1．2kb的特异片段,灵敏度实验显示该方法所需PCR模板DNA量为0．1073ng/ul在PCR的基础上的巢度PCR可以将灵敏度提高约10000倍,所需模板DNA仅为0．01073pg/ul,在甘薯丛枝病的检测中是一种快速,灵敏,可靠的方法。     

21三体综合症植入前诊断的临床前运用研究

探讨建立一个高效、稳定的21三体遗传病植入前诊断的方法,以染色体G显带核型分析为对照,取正常成人外周血单个淋巴细胞40枚、21三体患者外周血单个淋巴细胞40枚及单卵裂球20枚,采用荧光定量PCR技术同时扩增21号染色体上特异区域基因片段(DSCR)和12号染色体上管家基因(GAPDH)片断作内对照,结果在正常组织同时扩增二片断的有效率为95%(38/40),扩增产物的荧光强度比值为1.00±0.05;21三体患者单个淋巴细胞同时扩增二片断的有效扩增率为92.5%(37/40),DSCR/GAPDH荧光强度的比值约为1.58±0.17;单卵裂球的扩增效率为80.0%(16/20).三者实验结果与染色体核型分析结果完全一致,准确率100%.研究结果表明荧光定量PCR技术产前检测21三体综合征具有准确、快速、安全、实用等特点,有较高的临床推广应用价值.     

应用复合增强剂扩增人巨细胞病毒pp65全基因

在PCR过程中 ,模板GC含量过高是一个不利因素。如果设计扩增片段较长 ,则进一步增加了PCR扩增的难度。解决这一问题对于以PCR成功获取富含GC的长基因有非常重要的意义。以人巨细胞病毒pp65全基因 (约 1 95 0bp ,GC %为 67%)为例 ,在PCR系统中测试不同添加剂(甘油、乙醇、DMSO、甜菜碱等 )及各种组合 ,摸索扩增目的基因的最佳条件。结果发现 :无或单一的添加剂都不能获得目的基因片段 ,只有当同时使用DMSO和甜菜碱 ,并在适当浓度时才能够获得特异产物。在PCR系统中包含复合增强剂能有助于高GC %、长基因片段的扩增 ,为解决此类问题提供了一种有效的途径。     

家兔供体卵裂球细胞周期对核移植胚胎发育的影响

通过化学药物处理使家兔桑椹胚卵裂球的细胞周期分别同期化于G1、S或G2期 ,然后分别移入去核的MⅡ期卵母细胞中 ,以研究供体核细胞周期对家兔胚胎细胞核移植效率的影响。试验结果表明供体核细胞周期对家兔核移植胚胎的发育潜力有明显影响 ,以G1期卵裂球为供体的核移植重组胚的激活率、卵裂率、桑椹胚、囊胚和孵化囊胚率及囊胚平均细胞数分别为 87 3% (32 2 / 36 9)、 84 9% (2 99/ 35 2 )、 71 5 % (193/ 2 70 )、5 8 0 % (76 / 131)、 35 1% (4 6 / 131)和 12 6± 4 8,显著高于S期 [79 6 % (10 9/ 137)、 74 4% (93/ 12 5 )、30 3% (33/ 10 9)、19 3% (17/ 88)、 6 8% (6 / 88)和 118 8± 3 5 ]、G2期 [6 3 6 % (70 / 110 )、6 0 % (6 0 / 10 0 )、16 9% (11/ 6 5 )、 16 3% (7/ 43)、 4 7% (2 / 43)和 110 6± 5 8]和未经同期化处理的卵裂球 [78 1% (185 /2 37)、 73 2 % (15 8/ 2 16 )、 49 4% (4 3/ 87)、 32 2 % (2 8/ 87)、 12 6 % (11/ 87)和 12 0 5± 4 4](P <0 0 5 )。来源于G1期卵裂球的 144枚克隆胚胎移植到 12只受体中 ,6只妊娠并产下 19只活仔 ,产仔率为 13 2 % ,显著高于来源于S期 (5 8% ,7/ 12 0 )、G2期 (0 ,0 / 12 6 )或未同期化卵裂球的克隆胚胎 (5 3% ,8/ 15 0 ) (P <0     

西部马脑炎病毒基因组序列的半套式RT-PCR检测

为进一步提高RT－PCR检测西部马脑炎病毒（WEE）病毒基因组方法的敏感性,采用半套式PCR扩增病毒基因组特异序列,首先采用逆转录法将病毒基因组RNA逆转录为cDNA,然后以此cDNA为模板,进行扩增。对扩增后电泳检查无可见DNA条带的产物进行半套式PCR;与此同时对扩增的循环数、Mg^ 浓度和退火温度等条件进行了优化,以进一步提高扩增的特异性。结果第一轮PCR未扩出特异笥片段的WEE病毒稀释度,其半套式扩增出特定大小的DNA产物;同时优化的条件提高了扩增产物的特异性。扩增产物约为190bp的单一DNA片段,其大小与预期的相一致,结果表明采用半套式RT－PCR方法检测WEE病毒的基因组序列的敏感性可提高100倍以上。     

竞争性RT-PCR法及对大肠杆菌acrA基因mRNA表达水平的定量研究

建立了用于检测大肠杆菌(Escherichia coli)ATCC25922的acrA基因mRNA表达水平的定量竞争性RT-PCR(QC-RT-PCR)体系。PCR合成目标片段的突变型片段(321bp)作为内标准模板(Internal standard,IS),与目标片段一致的片段(389bp)作为目标模板(Target standard,TS),优化两种模板共扩增体系;梯度稀释IS与等量大肠杆菌cDNA样本共扩增,扫描电泳条带,软件分析数据。结果表明,引物设计合适,以IS和TS为模板实现共扩增,产物(321bp和389bp)通过1.5%琼脂糖凝胶电泳有效分离;梯度稀释IS与cDNA共扩增产物出现亮度梯度电泳条带;获得一元回归曲线y=-0.345 0.097x(相关系数r=0.959,标准差s=0.05997)。该研究成功构建内标准模板,优化的共扩增PCR体系实现了对大肠杆菌ATCC25922中acrA基因mRNA表达水平的检测,具有简便、高效、敏感度高等优点。     

抗胃癌免疫毒素表达质粒的构建、表达及对肿瘤细胞的特异杀伤

以含单链抗体 ( Sc Fv) 3H1 1基因全长的质粒 DNA为模板 ,利用 PCR技术扩增 3H1 1 Sc Fv基因片段 ,扩增片段及绿脓杆菌外毒素 PE38表达质粒 p YR39- 1 - PE38经 H ind /N de 酶切、连接 ,转化大肠杆菌 BL2 1,构建免疫毒素的表达质粒 p YR3H1 1 - PE38.转化菌在 IPTG诱导下 ,表达免疫毒素 3H1 1 - PE38,3H1 1 - PE38经纯化、变性、复性处理后 ,通过 MTT法检测其对胃癌细胞MGC80 3的杀伤活性 .结果表明 ,3H1 1 - PE38浓度不变 ,其杀伤率在一定的范围内随作用时间的延长而增加 ,当浓度为 5× 1 0 -8mol/L,作用时间为 60 h时 ,其对胃癌 MGC80 3细胞的杀伤率达74 .2 % ,而同等条件下抗 DNA免疫毒素 p Ig2 0 - PE38的杀伤率仅为 9.2 % ;作用时间一定 ( 60 h) ,免疫毒素浓度与杀伤率呈正相关 ,在 1 0 -10 mol/L以下 ,杀伤率几乎为零 ,而浓度高于 5× 1 0 -8mol/L时 ,杀伤率超过 70 % . 3H1 1 - PE38能够有效杀伤与之特异结合的胃癌细胞 ,具有潜在的应用前景     

棉铃虫细胞色素P450 cDNA片段的克隆与序列分析

以 5龄实验室敏感品系棉铃虫Helicoverpaarmigera的总RNA为模板 ,采用简并性引物 ,利用反转录 -多聚酶链式反应 (RT PCR)扩增出了 2个新的长度分别为 2 3 7bp和 2 40bp的cDNA片段。序列分析表明 ,2 3 7bp的cDNA片段与棉铃虫P45 0CYP4家族有较高的相似性 ,最高达 73 %;而 2 40bp的cDNA片段与CYP6家族有较高的相似性 ,最高达 49%。     

Rapid sexing of murine preimplantation embryos using a nested,multiplex polymerase chain reaction (PCR)

The objective of this study was to develop a rapid and efficient means of sexing murine preimplantation embryos at the 4- to 8-cell stage of development. To achieve this goal, a nested, multiplex polymerase chain reaction (PCR) was optimized using DNA from male and female mice and primers specific for X- (DXNds3)- and Y- (Sry,Zfy) gene sequences. Sensitivity of the assay was measured using groups of 4, 2, or 1 blastomere from dissociated embryos. Efficiency was evaluated using single blastomeres obtained by embryo biopsy. Accuracy of sexing was determined by comparing single-cell results with those of matched blastocysts. Robust amplification of male (XY) and female (XX) gene sequences was obtained in less than 6 hours. The percentage of male (3 bands) and female (1 band) reactions for groups of 4, 2, or 1 blastomere was 100% (6/6), 100% (15/15), and 94.4% (17/18), respectively. Assay efficiency for single, biopsied blastomeres from 4 to 8 cell embryos was 95.8% (207/216). For male and female embryos, sexing of single blastomeres accurately predicted results of matched blastocysts, 100% (10/10) and 100% (13/13), respectively. Simultaneous amplification of one X- and two Y-gene sequences ensured correct interpretation of sexing reactions. Short thermal cycling times and minimal tube handling increased the assay speed and decreased the potential risk of contamination. Mol. Reprod. Dev. 49:261–267, 1998. © 1998 Wiley-Liss, Inc.     

Sex determination of bovine embryo blastomeres by fluorogenic probes

One of the major challenges of using genetic information in marker assisted selection (MAS) is the detection of multiple marker loci from a small biopsy sample of a preimplantation stage embryo. The objective of this study was to develop a fast, nested, multiplex preamplification, polymerase chain reaction (PCR) method for the determination of sex in bovine embryo blastomeres. For this aim, ZFX/ZFY sequences were preamplified simultaneously with other genomic regions. The preamplification product was used as a template in an allelic discrimination assay, with nested primers and sex specific fluorogenic probes for ZFX and ZFY. Fluorogenic probes were used to eliminate the need for time consuming electrophoresis. Compared to sexing with Bovy/kappa-casein co-amplification method and other replicates from the same embryo, the accuracy of sexing with the use of fluorogenic probes after preamplification was 99% (112/113 blastomeres). The amplification efficiency was 96% (113/117 blastomeres).     

Sexing single bovine blastomeres using TSPY gene amplification

The testis-specific protein Y-encoded gene (TSPY) is a Y-specific gene present in variable copy number in many mammalian species, including cattle. We tested the applicability of the TSPY gene as a Y-specific marker to predict preimplantation embryo sex in Nelore (Bos indicus) cattle. Two blastomeres were removed from each embryo. A total of 36 single blastomeres and the remaining cells of their 18 matched in vitro conceived embryos were screened for TSPY amplification by nested-PCR. The results obtained from a single blastomere and the remaining cells of the same embryo were concordant in all cases. All blastomeres (16/16) from eight embryos produced with sexed sperm (specific for production of male embryos) were TSPY-positive. We conclude that TSPY is a good male-specific marker, the usefulness of which is probably enhanced by the high copy number. Other methods that are less time-consuming, such as real-time PCR, could be improved with the use of the TSPY gene sequences to generate primers and/or probes. This is the first report to demonstrate the applicability of the TSPY gene for sexing single cells in cattle.     

Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was <1 h. The pregnancy rate was 57.4% and all calves born were of the predicted sex (12 male and 21 female). Therefore, LAMP-based embryo sexing accurately determined gender and is suitable for field application.     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

Rapid sexing of water buffalo (Bubalus bubalis) embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The objective of this study was to identify a Y chromosome-specific sequence in water buffalo and to establish an efficient procedure for embryo sexing by LAMP. The homologues of a Y chromosome-specific sequence, bovine repeat Y-associated.2, in swamp and river buffalo were cloned, and designated swamp buffalo repeat Y-associated.2 and river buffalo repeat Y-associated.2, respectively. Sexing by LAMP was performed using primers for swamp buffalo repeat Y-associated.2. A 12S rRNA was also amplified by LAMP as a control reaction in both male and female. The minimal amount of the template DNA required for LAMP appeared to be 0.1-10 pg. The sensitivity was further examined using swamp buffalo fibroblasts as templates. When fibroblasts were lysed with NaOH, the minimal cell number required for detection of both male-specific and male-female common DNA appeared to be two cells, whereas correct determination of sex could not be achieved using fibroblasts lysed by heat denaturation. Embryo sexing was also performed using blastomeres from interspecies nuclear transfer embryos. The sex determined by LAMP for blastomeres corresponded with the sex of nuclear donor cells in analyses using four or five blastomeres as templates. The LAMP reaction required only about 45 min, and the total time for embryo sexing, including DNA extraction, was about 1 h. In conclusion, the present procedure without thermal cycling and electrophoresis was reliable and applicable for water buffalo embryos.     

Enhancing the mitotic index of blastomeres by thymidine synchronization

Mouse blastomeres were synchronized by exposure of the embryo to thymidine and colchicine. The mitosis index increased from 9% metaphases (colchicine synchronization) to 18% (thymidine + colchicine synchronization) per embryo. The in vitro development of embryos was not affected by treatment. This method appears to approach a level where successful sexing of embryos becomes possible.     

Sexing using single blastomere derived from IVF bovine embryos by fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) is a sensitive technique for molecular diagnosis of chromosomes on single cells and can be applied to sex determination of embryos. The objective has been to develop an accurate and reliable bovine Y chromosome-specific DNA probe in order to sex biopsed blastomeres derived from IVF bovine embryos by FISH. Bovine Y chromosome-specific PCR product derived from BtY2 sequences was labeled with biotin-16-dUTP (BtY2-L1 probe), and FISH was performed on karyoplasts of biopsed blastomeres and matched demi-embryos. Our FISH signal was clearly detected in nuclei of blastomeres of male embryos. FISH analysis of bovine embryos gave high reliability (96%) between biopsied blastomeres and matched demi-embryos. These results indicated that the BtY2-L1 bovine Y chromosome-specific FISH probe was an effective probe for bovine embryo sexing, and the FISH technique of probe detection could improve the efficiency and reliability.     

The effect of oxygen on the development of preimplantation mouse embryos in vitro

The optimal oxygen tension for development of preimplantation mouse embryos to the blastocyst stage in vitro was found to be between 2.5% and 5%. One- and two-cell embryos had a more sharply defined range of oxygen tension capable of supporting development than 8-cell and morula stages. At all stages of development, more embryos developed to the blastocyst stage under 5% O2 compared to the numbers of developing under higher oxygen tensions (20% and 40% O2). The blastocysts developing under 20% O2 had fewer blastomeres than those which developed under 5% O2. As the time required for development to the blastocyst stage in vitro increased, there were fewer blastomeres present at the blastocyst stage. These results indicate that the cleaving mouse embryo has an optimal oxygen requirement in vitro of about 5%. At higher oxygen tensions, fewer embryos develop to the blastocyst stage and in those which do develop, there are fewer cell divisions. If a gradient of oxygen tension exists across the blastomeres from the outside of the embryo to its centre, the blastomeres might be using this gradient to obtain imformation about their location within the embryo and respond accordingly. Thus blastomeres on the outside at a higher oxygen tension would divide at a slower rate and form trophectoderm whereas those on the inside at a lower oxygen tension would divide more rapidly and contribute to the inner cell mass.     

A rapid improved method for sexing embryo of water buffalo

The objective of the experiment of this paper is to develop and improve in the sexing method for preimplantation embryos of water buffalo (Bubalus bubalis) using loop-mediated isothermal amplification (LAMP) reaction. Embryo sexing has been recognized to control effectively the sex of offspring in the embryo transfer industry. A rapid and simple detection system was established by adding ethidium bromide (EB) or 5μl of CuSO4 (3M) to the product of LAMP reaction. The result of these additions after 2 min was a color change and a precipitate. It could be employed as an alternative method in the detection of the reaction products in place of the time consuming electrophoresis or the turbidity meter. The in vitro produced buffalo embryos were divided into one to eight pieces using a microblade attached to a micromanipulator. The cell number in each piece was counted before sexing. Sexing of DNA samples extracted from one to five biopsies cells was performed by LAMP. After biopsy, the remaining part of the embryos was used to confirm the sex by polymerase chain reaction (PCR). Fifty buffalo embryos were used and the accuracy of sex prediction was 100% when the blastomeres dissociated from a morula exceeds three. In conclusion, the present procedure without turbidity meter and electrophoresis was reliable and applicable for sexing the water buffalo embryos.