不同DNA提取方法对隐孢子虫卵囊PCR检测的影响

为了提高隐孢子虫PCR检测的敏感性和效率,采用10种基因组DNA提取方法,对隐孢子虫卵囊DNA进行提取,对提取的DNA进行nested PCR扩增。经过3次重复试验,结果显示,Chelex 100法、FTA试纸法和Wizard DNAClean-Up System试剂盒法敏感性最高,能够稳定地扩增出1×10^2个卵囊提取的DNA,适合隐孢子虫病分子流行病学调查时大量样品DNA的提取。     

干肉制品中霉菌PCR鉴定方法的建立

为了早期预防传统牛肉干发生霉变,建立了牛肉干中真菌PCR的鉴定方法,在特异性和敏感性方面对所建立的PCR方法进行了验证。其中特异性试验以霉变的鲜牛肉、酵母菌、乳酸球菌、乳酸杆菌、大肠杆菌为材料进行验证;敏感性试验以霉变的鲜牛肉为材料,提取其DNA并将其稀释成不同的浓度进行PCR验证。结果显示：特异性试验中仅霉菌样品在约300 bp处出现特异性条带,而其他样品在该处无特异性条带出现,表明具有良好的特异性;敏感性试验中,霉菌DNA原液、10倍稀释、100倍稀释、1 000倍稀释时均出现了约300 bp的特异性条带,即霉菌DNA模板稀释度为1 000倍（2 ng/μL）时仍对引物有敏感性。该试验为市场传统牛肉干真菌PCR检测工作提供了依据。     

牙釉质基因鉴定麇鹿性别

性别鉴定是调查野生种群雌雄性比的重要方法,对野生动物种群管理具有重要意义。而牙釉质(AMEL)基因在性染色体上具有较高的保守性,在鉴定动物性别方面得到了应用,本次试验采用北京麋鹿生态实验中心的15头糜鹿组织样本,其中11个来自雄性麇鹿鹿茸,4个来自雌性糜鹿静脉血液。对所取样本分别进行基因组DNA提取、AMEL基因片段PCR扩增、纯化、测序。得到了AMEL基因鉴定和实际雌雄性别个数差异不显著(P>0.05)。结果表明:雌性麋鹿产生1条322 bpX带和1条N带,雄性麋鹿则产生322 bpX带和277 bpY带以及1条N带,雄鹿(10/10)和雌鹿(4/4)性别鉴定结果分别都与实际性别符合,所以,使用鹿茸角和血液样本进行AMEL.基因扩增电泳分析可以对糜鹿性别鉴定。     

蚕微卫星DNA体外自我扩增条件初步研究

采用一种类似PCR的基因组自我引发PCR(GSP PRC)法 ,初步研究了家蚕、野桑蚕、天蚕、蓖麻蚕和柞蚕的微卫星DNA的体外基因组自我引发PCR扩增条件。质量浓度为 10 0mg/L的基因组DNA经 10 0℃变性 15min后 ,与等量相同浓度未变性的DNA混合作为模板 ,在 80 μL扩增体系 [含 0 2mmol/LdNTPs、5 0mmol/LKCl、10mmol/LTris HCl(pH 9 0 )、4mmol/LMgCl2 、1 2 5UTaqpolymerase]中加入 1μL此混合模板 ,在92℃、1min→ 5 5℃、2min→72℃、2min ,30～ 90次累积循环的扩增条件下 ,成功地得到了PCR产物。反应体系中 ,基因组DNA的适宜质量浓度为 1 2 5mg/L。GSP PCR产物经琼脂糖凝胶电泳后的部分片段回收后 ,在同样条件下 ,30～ 6 0个循环可得到同样大小范围的产物。GSP PCR产物经 6 %的变性测序胶电泳 ,得到典型的梯状带 ,表明为微卫星DNA。     

Comparison of four methods of extracting DNA from D. gallinae (Acari: Dermanyssidae)

Dermanyssus gallinae is one of the most serious ectoparasites of poultry and it has been implicated as a vector of several major pathogenic diseases. Molecular detection of such pathogens in mites is crucial and therefore, an important step is the extraction of their DNA from mites. So, we compared four DNA extraction protocols from engorged and unfed individual mites: a conventional method using a Cethyl Trimethyl Ammonium Bromide buffer (CTAB), a Chelex resin, a Qiamp DNA extraction kit and a more recent one filter-based technology (FTA). The DNA samples have been tested for their ability to be amplified by an amplification of a D. gallinae 16S rRNA gene region. The best results were obtained using CTAB and Qiagen methods at the same time with unfed and engorged mites (96% and 100% of amplified samples). FTA produced similar results when using unfed mites but not when processing engorged ones (96% and 70%). Finally, the Chelex method was the least efficient in terms of DNA amplification, especially when applied on engorged individuals (50%). The possible inhibitor role of these Chelex extracted DNA was demonstrated by the means of a PCR control on PUC plasmid. No difference was observed with CTAB, Qiamp DNA extraction kit or FTA methods using DNA extracted one year before.     

牛传染性鼻气管炎病毒内蒙古分离株gG基因的PCR扩增

参考牛传染性鼻气管炎病毒全基因序列(GenBank)设计1对特异性引物,以牛传染性鼻气管炎病毒内蒙古分离株提取的总DNA为模板,运用PCR方法成功地扩增出牛传染性鼻气管炎病毒内蒙古分离株gG基因,并用琼脂糖凝胶电泳检测扩增产物。     

Diagnosis of Canine Brucellosis: Comparison between Serological and Microbiological Tests and a PCR Based on Primers to 16S-23S rDNA Interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol–chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.     

A PCR-based sex determination method for possible application in caprine gender selection by simultaneous amplification of the Sry and Aml-X genes

Sex determination of livestock is performed to achieve the objectives of livestock breeding programmes. Techniques for sex determination have evolved from karyotyping to detecting Y-specific antigens and recently to the polymerase chain reaction (PCR), which appears to be the most sensitive, accurate, rapid and reliable method to date. In this study, a PCR-based sex determination method for potential application in goat breeding programmes was developed. Primers were designed to amplify a portion of the X amelogenin gene (Aml-X) on the X chromosome to give a 300 bp product and Sry gene on the Y chromosome to give a 116 bp product. PCR optimization was performed using DNA template extracted from a whole blood sample of Jermasia goats (German Fawn x Katjang) of both sexes. It was possible to identify the sex chromosomes by amplifying both male- and female-specific genes simultaneously in a duplex reaction with males yielding two bands and females yielding one band. The Aml-X primer set, which served as an internal control primer, did not interfere with amplification of the Y-specific sequence even when a low amount of DNA (1 ng) was used. The duplex reaction subjected to a blind test showed 100% (14/14) concordance, proving its accuracy and reliability. The primer sets used were found to be highly specific and were suitable for gender selection of goats.     

猪性控精子纯度实时荧光定量PCR检测方法的建立

研究旨在利用实时荧光定量PCR法检测杜洛克猪性控精子，以期建立一种快速、准确且经济高效的性控精子纯度检测方法。选取位于猪Y染色体上的Y染色体性别决定区（sex-determining region Y，SRY）基因和位于X染色体上的A-激酶锚定蛋白4（A-kinase anchoring protein 4，AKAP4）基因，以猪耳组织样提取的基因组DNA为模板进行PCR扩增验证引物的特异性，利用胶回收试剂盒进行回收并质粒小提，将获得的两种质粒经检测后稀释至相同浓度（20 ng/μL），混合构建含有不同比例SRY和AKAP4基因的质粒模板，用于绘制检测精子纯度所用标准曲线的反应模板。分别使用SRY和AKAP4基因特异引物检测3个混合的X精子（P.x_gro1、P.x_gro2、P.x_gro3）和3个混合的Y精子（P.y_gro1、P.y_gro2、P.y_gro3）的纯度。结果显示，用SRY基因特异性引物检测P.x_gro1、P.x_gro2、P.x_gro3精子的纯度分别为91.44%、91.93%、88.99%，P.y_gro1、P.y_gro2、P.y_gro3精子的纯度分别为89.91%、87.31%、88.71%；用AKAP4基因特异性引物检测P.x_gro1、P.x_gro2、P.x_gro3精子的纯度分别为91.44%、91.93%、88.99%，P.y_gro1、P.y_gro2、P.y_gro3精子的纯度分别为89.91%、87.31%、88.71%；卡方适合性检验结果显示，两次检测结果之间差异不显著，表明使用这种方法进行精子纯度检测所得结果准确。本试验通过实时荧光定量PCR法准确检测了经流式细胞仪分选后的精子的纯度，建立了一种利用实时荧光定量PCR法检测猪精液中X精子和Y精子比例的新方法。     

Sexing of newly-hatched chicks using DNA isolated from chorio-allantoic membrane samples by polymerase chain reaction in Denizli chicken

1. The aim of the present study was to determine the sex of newly-hatched chicks of Denizli chicken, a local Turkish breed, by polymerase chain reaction (PCR) using DNA extracted from the chorioallantoic membrane (CAM). 2. Fertilised eggs were incubated individually and a total of 20 CAM samples were collected following the hatching process. DNA was isolated from the CAM samples and PCR was performed using W-repeat (W) and 18 S ribosomal gene (R) primers. 3. Screening of the PCR products by agarose gel electrophoresis revealed that males have a single band (256 bp) and females have an extra second band (415 bp) as expected. 4. The present study describes a reliable, rapid, and simple multiplex PCR protocol that can be put into use to sex local breeds of chicken in which phenotypic sexing is impossible, using DNA isolated from the CAM that is discarded and remains attached to the egg shell following the hatching process.     

禽传染性支气管炎DNA疫苗在鸡体内的分布和安全性

采用本实验室构建的3种禽传染性支气管炎病毒基因的真核表达质粒pIBVS1、pIBVM、pIBVN,按各50 mg/L配制成质量浓度为150 mg/L的DNA疫苗,经腿部肌肉分点注射免疫1周龄SPF雏鸡。分别在免疫后24 h,73、0及60d,采集试验鸡的血液、心、肝、脾、肺、肾、胸腺、性腺（卵巢/睾丸）及注射部位肌肉进行组织总DNA抽提,以组织总DNA为模板进行PCR扩增。另外,在对照组DNA模板中加入不同拷贝数的质粒,确定PCR反应的灵敏性。以纯化后的组织总DNA为模板,PCR法检测质粒DNA整合到鸡细胞染色体基因组上的可能性,评价疫苗的安全性。结果表明,该疫苗在24 h内迅速分布于全身,并能在血液及所有检测的组织器官内持续分布60 d;纯化后的组织基因组DNA经PCR扩增均呈阴性,未发现整合现象,证实该DNA疫苗的安全性好。     

Isolation of genomic DNA from feathers.

The use of feathers in veterinary clinical practice simplifies the sampling of avian genomic DNA, especially when blood extraction is difficult because of the age or the size of the bird. A rapid and accurate protocol was used to isolate high-quality genomic DNA from feathers. The technique includes a lysis step of the feather quill, which differs in temperature and time of incubation depending on the feather size. Purification of genomic DNA is performed with phenol: chloroform: isoamyl alcohol extraction and ethanol precipitation. This protocol consistently provided significant amounts of high-quality genomic DNA from more than 800 birds belonging to 120 different species. Genomic DNA isolated with this method was used for Southern blotting and also in several polymerase chain reaction systems devoted to sex determination and paternity testing.     

Sensitivity of different Trypanosoma vivax specific primers for the diagnosis of livestock trypanosomosis using different DNA extraction methods

There are several T. vivax specific primers developed for PCR diagnosis. Most of these primers were validated under different DNA extraction methods and study designs leading to heterogeneity of results. The objective of the present study was to validate PCR as a diagnostic test for T. vivax trypanosomosis by means of determining the test sensitivity of different published specific primers with different sample preparations. Four different DNA extraction methods were used to test the sensitivity of PCR with four different primer sets. DNA was extracted directly from whole blood samples, blood dried on filter papers or blood dried on FTA cards. The results showed that the sensitivity of PCR with each primer set was highly dependant of the sample preparation and DNA extraction method. The highest sensitivities for all the primers tested were determined using DNA extracted from whole blood samples, while the lowest sensitivities were obtained when DNA was extracted from filter paper preparations. To conclude, the obtained results are discussed and a protocol for diagnosis and surveillance for T. vivax trypanosomosis is recommended.     

Sex identification of Japanese black bear,Ursus thibetanus japonicus,by PCR based on amelogenin gene

A method for sex identification of the Japanese black bear was examined using a polymerase chain reaction (PCR) and sequencing of a part of the amelogenin gene. This gene is located on the X and Y chromosomes, and there are 54 nucleotide deletions on the Y chromosome-specific gene. Forty-seven (26 male and 21 female) DNA samples and 23 (13 male and 10 female) DNA samples, respectively extracted from white blood cells and hairs of Japanese black bears were analyzed. The primers SE47 and SE48 from this X-Y homologous region were used in sex identification by PCR amplification. These primers amplified X- and Y-specific bands, which could be used to discriminate between sexes by a length polymorphism in all samples. We suggest that PCR amplification using the primers SE47 and SE48 is useful for sex determination of the Japanese black bear and could be applied to DNA analysis of small samples such as hairs.     

Ostrich (<Emphasis Type="Italic">Struthio camelus</Emphasis>) production in Egypt

This review discusses the historical, developmental and practices of ostrich farming in Egypt. In the early 20th century, ostrich farming was very important for production of ostrich feathers and documents were produced to perfect the art of procuring the plumes from the birds and subsequently processing them. Pharaohs used ostrich feathers for adornment. Of 43 provinces, 12 were featured in 2003-2004 as farming ostriches: Alexandria, Al-Behera, Al-Dakahlia, Al-Wadi Al-Gadid, Aswan, Cairo, El-Sharkia, Geiza, Ismailia, Kafr-El-Sheikh, Matrouh and Nubaria. Abattoirs and tanneries specialising in ostrich handling are limited to two. Egypt has numerous strengths and opportunities to develop its ostrich sector. Rising meat prices suggest that fresh ostrich meat is unaffordable to many locals. Funds may be allocated to local advertising campaigns to promote ostrich meat; provision of incentives to farmers; and improving the capacity of abattoirs.     

微量全血PCR技术鉴定雏鸡及鸡胚性别

为了便于常规PCR技术在鸡性别鉴定上的推广和应用,本研究以成年家鸡全血为模板,应用常规PCR反应缓冲液和Tap DNA聚合酶直接进行PCR扩增,对50 μL PCR反应体系中的血样(0.05~4.0 μL)和Tap DNA聚合酶的使用量(0.05~1.5 μL),以及循环次数(30~40)进行了优化,在此基础上对血样的抗凝剂和保存温度进行了探讨,并对62只1日龄雏鸡、80枚12日龄和80枚16日龄鸡胚的血样直接PCR进行了性别鉴定。结果表明,采集血样时可以使用ACD、肝素或EDTA作为抗凝剂,血样可以在4、－20或－80 ℃至少保存3个月;使用0.1 μL全血就能完成雏鸡和鸡胚性别鉴定,全血PCR鉴定性别与性腺性别的符合度为100%。与常规PCR相比,全血PCR节约了检测成本,提高了检测效率,减少了交叉污染可能。     

Molecular identification of encephalitozoon hellem in an ostrich

Microsporidia are obligate, intracellular, eukaryotic parasites found in a wide variety of vertebrate and invertebrate hosts. Scientific literature contains a small number of reports of these parasites in psittacine hosts, and recently microsporidiosis was reported in the first nonpsittacine host, an ostrich. DNA was extracted from formalin-fixed, paraffin-embedded ostrich tissues, and a portion of the small subunit ribosomal RNA gene was sequenced to identify the microsporidian species. The organisms were identified as Encephalitozoon hellem, a parasite species that was first described in immunocompromised humans and recently reported in three psittacine species.     

Semen Collection and Polymerase Chain Reaction‐Based Sex Determination of Black‐Headed and Straw‐Necked Ibis

This study aimed to develop a polymerase chain reaction (PCR)‐based sexing and effective semen collection methods for black‐headed and straw‐necked ibis species. However, most birds are not sexually dimorphic, that is, the sexes appear similar. Therefore, the gender should be determined before semen collection. DNA was extracted from the blood samples of 11 black‐headed and 4 straw‐necked ibis. The sex was determined after PCR amplification of the EE0.6 region of W‐chromosome. The PCR products were separated using gel electrophoresis. A single band indicated the presence of the EE0.6 region and that the individual was a female, while no band indicated that the individual was a male. Further, the single bands from seven specimens were amplified. Semen collection was performed by massage or a combination of massage with electro‐ejaculation and was attempted during all four seasons. The semen was successfully collected in March from male straw‐necked ibis using the massage method. Limited motility, viability and concentration of straw‐necked ibis sperm were observed. The sperm length was 180 μm and that of the nucleus was 30 μm with acrosome located at the tip of the nucleus. Thus, the PCR‐based sexing proved to be an accurate molecular sexing method for black‐headed and straw‐necked ibis. Furthermore, we successfully collected semen and observed the stained sperm nucleus and acrosome of the straw‐necked ibis sperm. We propose that the use of this PCR methodology can be applied as a routine method for sex determination and semen collection in ibis species for future ecological research. However, considering our limited success, further studies on semen collection method are required.     

应用PCR技术检测柞蚕微孢子虫

采用斑迹抽提法提取柞蚕微孢子虫(Nosema pernyi)基因组DNA,基因组DNA的琼脂糖凝胶电泳图谱中有大小约15kb的清晰、完整条带。选用已报道的微粒子属16S rRNA基因的保守序列设计P1/P2和N1/N22对引物,对柞蚕微孢子虫基因组DNA进行PCR扩增,结果2对引物分别扩增出1条大小不同的特异谱条带,其中用引物N1/N2扩增可检测出0.47ng的DNA模板。应用同样的PCR引物、体系和反应条件,可有效扩增出受感染柞蚕幼虫、成虫中的微孢子虫基因组DNA条带。该项检测技术有望应用于柞蚕微粒子病的早期诊断。     

Sexing of pre-implantation ovine embryos through polymerase chain reaction-based amplification of GAPDH,SRY and AMEL genes

The ability to identify the sex of embryo and control of sex ratio has a great commercial importance to livestock industry. Prediction of embryonic sex could be useful in the management decisions of sex selection in breeding programs. Several methods have been attempted to determine the sex but the polymerase chain reaction (PCR)-based sexing method is generally favoured, as it is cost effective, simple and reliable. The aim of the present study was to identify sex of sheep embryos produced in vitro through amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sex-determining region Y (SRY) and amelogenin genes present in genomic DNA (gDNA) of embryos through PCR. To avoid false interpretation of the result by no amplification of SRY in female embryos, a duplex PCR was approached to amplify combinedly SRY and GAPDH genes. Sex-specific blood was used in PCR as positive control. In vitro sheep embryos were produced as per standardized protocol of laboratory. Sexing of sex-specific blood and in vitro produced embryos were approached though PCR to amplify the respective genes using gDNA present in the sample without its traditional isolation. The accuracy of sex prediction for embryos was 100% by this procedure.