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通过对互助猪合成母系白系的36头猪采血样、DNA提取、PCR扩增、产物电泳及产物酶基因型进行判定.结果表明,采用PCR酶切技术能快速、准确地从DNA水平上鉴别氟烷基因型,并且鉴别互助猪母系的氟烷基因型基本上属于氟烷敏感基因隐型. 相似文献
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猪氟烷基因PCR-RFLP检测新引物设计与条件优化 总被引：1，自引：0，他引：1
本文在测定4个不同品种猪包含突变位点的氟烷基因序列的基础上,重新设计了PCR引物,并对反应体系和反应条件进行了优化。实验结果表明:新设计的引物在PCR扩增中对DNA模板量要求不高,PCR反应对温度变化不敏感,具有较高的稳定性,结合限制性酶切技术,能快速准确地鉴别猪氟烷基因的三种基因型,可应用于猪氟烷基因的分子检测。 相似文献
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《中国畜牧兽医》2010,(7)
本试验选择了103头经过30多年趋异选择的皮特兰猪和大白猪,检测了其PIT1基因的多态性,并对其父系和母系遗传的基因频率进行了比较。对试验猪采集血样,提取基因组DNA,并经PCR扩增、RFLP基因分型及限制性内切酶RsaⅠ鉴定。试验数据用卡方检验进行统计分析。结果表明,AB型个体占57.3%,其中大白猪父系占26.2%,皮特兰猪父系占18.5%,皮特兰猪母系占12.6%;AA基因型个体占20.4%,其中大白猪占7.8%,皮特兰猪父系占4.8%,皮特兰猪母系占7.8%;BB型个体占22.3%,其中大白猪父系占6.8%,皮特兰猪父系占9.7%,皮特兰猪母系占5.8%。A、B的等位基因频率分别为49.0%、51.0%。通过对皮特兰猪母系和父系遗传进行研究,结果表明,PIT1基因的表达在父系遗传中较为稳定;与母系遗传相比,父系遗传的B等位基因与体脂率降低和瘦肉率升高有一定的相关性,而皮特兰猪和大白猪父系遗传的B等位基因频率无明显差异。 相似文献
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三江白猪氟烷基因的基因频率分析 总被引：2，自引：0，他引：2
本试验随机抽取216头30日龄左右的三江白猪,从外周血液中提取基因组DNA,经纯化后应用所设计的引物进行PCR扩增,酶切鉴定后分析三江白猪氟烷基因(RYR1)的基因与基因型频率。结果表明:PCR反应条件优化后,扩增出659 bp的特异性基因片段,经HhaⅠ和HgiAⅠ两种内切酶消化后,发现三江白猪T/T(Haln/Haln)基因型频率为4.16%;C/C(HalN/HalN)型为88.43%;C/T(HalN/Haln)型为7.41%。HalN基因频率为92.13%,Haln基因频率为7.87%。此结果表明,三江白猪中氟烷敏感基因频率和基因型频率均较低,可以通过标记辅助选择方法很快予以剔除。 相似文献
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三江白猪氟烷基因频率分析 总被引：1，自引：0，他引：1
本试验随机抽取216头30日龄左右的三江白猪,从外周血液中提取基因组DNA,经纯化后应用所设计的引物进行PCR扩增,酶切鉴定后分析三江白猪氟烷基因(RYR1)的基因与基因型频率.结果表明PCR反应条件优化后,扩增出659 bp的特异性基因片段,经Hha Ⅰ和HgiAⅠ两种内切酶消化后,发现三江白猪T/T(Haln/Hal)基因型频率为4.16%;C/C(HalN/HalN)型为88.43%;C/T(HalN/Haln)型为7.41%.HalN基因频率为92.13%,Haln基因频率为7.87%.此结果表明,三江白猪中氟烷敏感基因频率和基因型频率均较低,可以通过标记辅助选择方法很快予以剔除. 相似文献
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猪应激综合症由氟烷基因型决定,有三种基因型,阳性,阴性和杂合子型产生应激综合征。采用聚合酶链反应（ＰＣＲ）扩增一段含有突变位蹼的氟烷基因片段,用限制性内切酶酶切分析,测定杜洛克猪品系３７头,有３头为杂合子,３４头为阴性。 相似文献
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通过猪毛检测丹麦长白猪氟烷基因的变异 总被引：11，自引：2，他引：9
采集５６头丹麦长白猪（其中１头为１周龄的仔猪，２６头种公猪，２９头种母猪）猪毛样品，粗提ＤＮＡ，进行ＰＣＲ扩增，得到６５９ｈｐ在氟烷基因中（即兰尼定受体基因）中第１８４３个碱基的异片段，经过ＨｈａＩ酶切和电泳来检测氟烷基的变异，即其基因型，结果表明，（１）５６头长白猪中，阳性猪（ｎ／ｎ）１头占１．７９％，杂合子猪（Ｎ／ｎ）为１８头，占３２．１４％，阴性猪（Ｎ／Ｎ）为３７头，占６６．０７％杂合子数实 相似文献
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Development of a diagnostic PCR assay based on novel DNA sequences for the detection of Mycoplasma suis (Eperythrozoon suis) in porcine blood 总被引：34，自引：0，他引：34
An efficient method of control of porcine eperythrozoonosis (PE) caused by Mycoplasma suis is eradication of infection by detection and removal of infected carrier animals. At present, only a few tests are available for the diagnosis of these latent M. suis infections in pigs. The objective of this study was to develop a PCR assay based on novel DNA sequences for the identification of M. suis-infected pigs. A 1.8 kb EcoRI DNA fragment of the M. suis genome was isolated from the blood of pigs experimentally infected with M. suis. Specificity of the DNA fragment was confirmed by DNA sequence analysis and PCR using primers directed against sequences contained in the 1.8 kb fragment. PCR products of 782 bp in size were amplified only from M. suis particles prepared from the blood of experimentally infected pigs but not from any controls, comprising blood from gnotobiotic piglets and a panel of bacteria including other porcine mycoplasmas. PCR results were confirmed by dot blot hybridisation. The applicability of the PCR assay to diagnose M. suis infections in pigs was evaluated by investigating blood samples from 10 symptomatic pigs with clinical signs typical of porcine eperythrozoonosis and blood samples from 10 healthy pigs. The M. suis-specific PCR product was amplified from all samples taken at episodes of acute disease as well as from samples taken during the latent stage of infection, thus demonstrating the suitability of the PCR assay for detecting latent infected carrier animals. 相似文献
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以猪弓形虫核糖体DNA第一内转录间隔区(ITS1)序列为模板自行设计引物,建立了猪弓形虫病特异PCR诊断方法,并从弓形虫国际标准强毒株RH速殖子和疑似T.gondii感染猪全血及肺脏组织样品基因组DNA中扩增出了预期长度273bp的目的DNA片段。敏感性和特异性试验结果显示,该PCR方法能检测到的最低DNA量为0.001ng,且与相关的9种对照寄生虫、细菌和病毒无交叉反应。用建立的PCR诊断方法对临床30份猪弓形虫疑似病料和60份健康猪抗凝全血样品进行检测,结果30份病料中有24份呈现阳性;60份健康猪血中有5份为阳性;随机取两个临床样品的阳性PCR扩增片段进行克隆测序表明,二者序列与Gen-Bank中已登录的猪弓形虫ITS1基因相应部分序列完全相同。以上表明所建立的PCR方法具有高度的敏感性和特异性;本研究为猪弓形虫病的快速诊断提供了一种新方法。 相似文献
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During monitoring of certified pseudorabies (PRV)-free herds to confirm their PRV -free status, occasional individual gE-seropositive pigs are detected. These single-reactor pigs remain gE-seropositive when further serum samples are collected and tested. For the eradication programme to proceed, it is important to determine whether these pigs are only false positives or are; in fact, infected with field PRV. The purpose of this study was to determine whether the polymerase chain reaction (PCR) could detect field PRVDNA in single-reactor pigs and so confirm positive reactions in the serologic monitoring programme. First, DNA samples of various tissues from 15 single-reactor pigs all from different herds were examined for field PRV by PCR. Additionally, serum samples from these pigs were analyzed in a gE-confirmation enzyme linked immunosorbent assay (gE-confirmation ELISA). PCR detected PRVDNA in five of the 15 pigs, and these results were confirmed by the gE-confirmation ELISA. The remaining 10 pigs that tested negative in the PCR also tested negative in the gE- confirmation ELISA. We conclude that PCR can be used to discriminate between true and false serological positive single-reactor pigs and, moreover, that the gE-confirmation ELISA confirms these PCR results. 相似文献
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The course of naturally acquired Lawsonia intracellularis infection was studied in 41 pigs by testing blood and faeces samples collected four to seven times from before weaning to slaughter 5 months old. At slaughter, a sample of ileum was taken for histopathology. In the first sampling when the pigs were 2-4 weeks old maternally derived IgG against L. intracellularis was demonstrated by immunofluorescence antibody test in nine pigs whereas the bacterium was detected by PCR in faeces from six pigs. The maternally derived antibodies did not prevent pigs from becoming infected as seven pigs later on shed and/or were seropositive for L. intracellularis. The lowest prevalence of L. intracellularis was observed in 6-13 weeks old pigs and it seemed as though L. intracellularis in early infected pigs only activates a minor antibody response. At slaughter 66% of the pigs were found positive by immunofluorescence antibody test compared to 24% by immunohistochemistry on ileal samples. Thus, applied at the time of slaughter the antibody test appeared to be a highly sensitive ante-mortem diagnostic tool for identifying L. intracellularis exposed pigs with or without current proliferative enteropathy. 相似文献
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猪附红细胞体PCR诊断方法的建立及应用 总被引：1，自引：0，他引：1
根据基因库中已登录的猪附红细胞体新的基因组DNA序列设计引物,从疑似猪附红细胞体(Mycoplasma suis)感染猪全血样品基因组DNA中扩增出了预期长度666 bp的目的DNA片段,通过对24份临床疑似病例样品的PCR扩增及其他病原微生物基因组DNA的特异性扩增试验,建立了E.suis的PCR诊断方法;进一步对PCR产物克隆、测序分析表明,与国外已报道的AJ504999株相关区域核苷酸、氨基酸序列同源性分别为98.19%和96.85%,表明我国分离株与国外株基因组存在差异。 相似文献
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Magdalena Jacobson Stina Englund András Ballagi-Pordány 《Journal of veterinary diagnostic investigation》2003,15(3):268-273
Lawsonia intracellularis is an intracellular organism that causes proliferative enteritis in pigs. This bacterium is difficult to culture, and antemortem demonstration of the microbe is therefore often performed on fecal samples by polymerase chain reaction (PCR). Polymerase chain reaction is sensitive and specific, but inhibitory factors in feces might cause false-negative results. This article describes the construction and use of an internal standard, a mimic. The mimic is amplified by the same primers as those used for L. intracellularis DNA and thus could indicate false-negative results in clinical samples. The amplicon was clearly visible when as few as 10 mimic molecules were added per amplification reaction and when no inhibitors werepresent. When fecal samples were spiked with the mimic, the detection limit was 10(2) molecules per PCR. Sixty clinical samples, 20 from wild boars, 20 from growing pigs with diarrhea, and 20 from pigs without diarrhea, were prepared by a boiling procedure and subjected to PCR together with 10(3) mimic molecules. Nine samples were positive, of which 7 originated from pigs with diarrhea and 2 from pigs without diarrhea. In 14 samples from wild boars, in 8 samples from pigs without diarrhea, and in 3 samples from pigs with diarrhea, neither the mimic nor the target DNA was visible. This indicated the presence of inhibitors in these samples. It is concluded that the mimic can be used as an internal control in the diagnosis of L. intracellularis to indicate inhibition of PCR. 相似文献
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Shibata I Okuda Y Yazawa S Ono M Sasaki T Itagaki M Nakajima N Okabe Y Hidejima I 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2003,65(3):405-408
Porcine circovirus type 2 (PCV2) shedding patterns were investigated by polymerase chain reaction (PCR) for the detection of PCV2 DNA, and the diagnostic suitability of a sample for the PCR was examined by using different types of samples. In the experimental infection, sixteen pigs were inoculated intranasally with PCV2. The samples, including oropharyngeal and nasal swabs, feces, whole blood and serum became positive for PCV2 DNA by PCR immediately after the inoculation, and almost all samples remained positive during the observation period, post-inoculation-day 70. Field samples were collected from 313 pigs in five different age groups. The overall percentages of positive samples in the whole blood, nasal swabs, and feces detected by PCR were 30.4%, 19.2%, and 20.4%, respectively. The frequency of positive samples increased after the nursery stages and reached a peak in the 3 to 4-month-old pigs. These results indicate that PCV2 infection may occur after weaning, that PCV2 DNA may be present in whole blood for a long period after infection, and that whole blood and serum are the most suitable sample types for the PCR analysis of PCV2. 相似文献
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Gisele S. Porto Raquel A. Leme Alais M. Dall Agnol Tatiana C.G.D. de Souza Amauri A. Alfieri Alice F. Alfieri 《Journal of veterinary science (Suw?n-si, Korea)》2021,22(6)
BackgroundSuid gammaherpesvirus 3, 4, and 5 (porcine lymphotropic herpesvirus – PLHV-1, -2, and -3) are viruses that infect domestic and feral pigs.ObjectivesThis study examined the presence of PLHV DNA in biological samples from free-living wild boars circulating in a Brazilian geographical region with a high density of commercial domestic pigs.MethodsLung samples of 50 free-living wild boars were collected by exotic wildlife controller agents between 2017 and 2019 in the state of Paraná, southern Brazil. Lung and spleen fragments were obtained from six fetuses collected by hysterectomy post mortem from a pregnant sow. A polymerase chain reaction (PCR) assay using consensus primers (pan-herpesviruses) was performed to detect PLHV DNA. The samples showing positive results for PLHV DNA were submitted to single-round PCR assays with the specific primers for identifying PLHV-1 (213-S/215-As), PLHV-2 (208-S/212-As), and PLHV-3 (886s/886As). The specificity of the species-specific PCR products was assessed by nucleotide sequencing of the amplicons.ResultsForty-eight (96%) of the 50 lung samples analyzed were positive for PLHV by PCR using pan-herpesvirus primers. In 33 (68.75%) of the positive samples, at least two PLHV species were identified simultaneously. The DNA of PLHV-1, -2, and -3 was found in free-living wild boars of all ages, but not in the fetuses, even though they were from a sow that tested positive for all three viruses.ConclusionThese viruses are endemic to the population of feral pigs in the Brazilian region evaluated, as well as in domesticated pigs. 相似文献