转基因牛多重PCR检测方法的建立

为建立一种快速检测转基因牛的多重PCR方法,针对牛线粒体16 S rRNA基因(BOS mtD-NA16 S rRNA,BOS)、转基因动物常用标记基因新霉素磷酸转移酶基因(NPT Ⅱ)、人乳铁蛋白编码基因(human laetoferrin gene,hLF)、人-α-乳清蛋白编码基因(human α-lactalb...     

一株猪细小病毒7群的鉴定和分离

猪细小病毒7群(Porcine parvovirus 7,PPV-7)是2016年首次从美国猪群发现和鉴定的新的猪细小病毒。为弄清我国猪群中是否存在PPV-7,本文用PPV-7特异性的实时荧光定量PCR和常规PCR方法对采集的10份猪流产胎儿和10份保育仔猪病料分别进行检测,结果发现其中1份保育仔猪样品的常规PCR和实时荧光定量PCR检测结果均为阳性,常规PCR扩增出的246 bp特异性条带测序和分子遗传演化时发现,该序列与PPV-7参考毒株KU5637332和KY996757的同源性分别为99.6%和98.0%,表明该样品中含有PPV-7,进一步用PK-15细胞分离病毒,连续传代5次后PK-15细胞都没有出现典型的细胞病变,但其上清液用实时荧光定量PCR方法都可以检测到该病毒。本研究结果证实我国猪群中存在PPV-7的感染,且检测到的PPV-7毒株能在PK15细胞中增殖。     

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR

Johne’s disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.     

A Qualitative Study of State‐Level Zoonotic Disease Surveillance in New England

Zoonotic diseases are infectious diseases transmittable between animals and humans and outbreaks of these diseases in animals can signify that humans are also infected (or vice versa). Thus, communication between animal and human health agencies is critical for surveillance. Understanding how these agencies conduct surveillance and share information is important for the development of successful automated zoonotic monitoring systems. Individual interviews were conducted with 13 professionals who perform animal or human zoonotic disease surveillance in one of the New England states. Questions centred on existing surveillance methods, collaborations between animal and human health agencies, and technological and data needs. The results showed that agencies routinely communicate over suspected zoonotic disease cases, yet there are barriers preventing automated electronic linking of health data of animals and humans. These include technological barriers and barriers due to sensitivity and confidentiality of information. Addressing these will facilitate the development of electronic systems for integrating animal and human zoonotic disease surveillance data.     

应用PCR技术检测鹅细小病毒

根据鹅细小病毒GPVH1株结构蛋白VP1与VP3编码基因非重叠核苷酸序列设计了一对引物GPR1 /GPR2 ,用这对引物对感染器官内的GPR和接种鹅胚增殖的GPV进行PCR扩增 ,其结果引物GRP1 /GPR2能特异性地扩增GPVVP1 VP3区段核苷酸序列 ,扩增出与设计核苷酸片段大小相符的 0 6kb的序列 ,而对照的鹅副粘病毒cDNA及鸭瘟病毒 (DPR)DNA对照组均出现阴性结果。     

Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR

A diagnostic procedure, based on a polymerase chain reaction method (PCR) was developed to detect infection of crayfish with the Oomycete Aphanomyces astaci. A set of oligonucleotide primers was designed to specifically amplify A. astaci DNA in the ITS region surrounding the 5.8S rDNA gene. The PCR amplifies a 115 bp amplicon. The specificity of the primers was demonstrated by testing on 27 A. astaci strains and against 20 non-A. astaci Oomycetes and 5 fungal species. Most of the non-A. astaci Oomycete or fungal species included in the study are either known parasites of freshwater crayfish cuticle or can be found in their natural environment. Specificity was also tested against crayfish tissue and some known parasites and bacteria infecting crayfish.

A protocol for the extraction of A. astaci DNA from infected crayfish tissue was developed. The optimised method allows the detection of two genome equivalents of purified A. astaci genomic DNA.

The method was tested on noble crayfish (Astacus astacus), artificially infected with A. astaci. Detection of A. astaci was possible at the very first time of sampling, which was 2 days after the beginning of spore exposure.     

套式PCR在猪流行性腹泻病毒感染病例诊断中的应用

猪流行性腹泻是对养猪业危害较大的一种疾病,导致腹泻的原因多种多样,给临床诊断带来了困难。本研究将套式PCR技术成功应用于猪流行性腹泻临床病例的快速诊断中,旨在为猪流行性腹泻临床病例的快速鉴别诊断提供一种新的思路。     

斯氏艾美耳球虫ITS1-5.8S rRNA-ITS2序列的克隆及PCR检测方法的建立

通过对多种鸡球虫和松鼠球虫18SrRNA和28SrRNA进行序列比对分析,在18SrRNA 3′端和28SrRNA 5′端保守区设计艾美耳属通用引物,以斯氏艾美耳球虫洛阳分离株LY卵囊基因组DNA为模板首次成功克隆到斯氏艾美耳球虫完整的ITS1-5.8SrRNA-ITS2序列,其大小为1 178bp,其中ITS1序列长度为423bp,5.8SrRNA为155bp,ITS2为600bp,斯氏艾美耳球虫LY株ITS1/2序列高度变异,与鸡球虫、啮齿动物球虫的序列相似性低于60%。然后在斯氏艾美耳球虫ITS1/2序列超变区设计种特异引物,建立了灵敏、特异的PCR检测方法。本研究结果将为兔球虫强致病种的临床诊断和揭示兔球虫种群遗传特征提供有效的分子工具。     

牦牛病毒性腹泻病PCR诊断方法的建立

采用阻断ELISA方法对来自天峻县的327份牦牛血清进行了牛BVDV特异性抗体的检测,共检出231份血清阳性,血清阳性率为70.64%。其中:在135份野牦牛血清中检出110份阳性血清,阳性率为81.48%;在192份家牦牛血清中检出阳性121份,阳性率为63.02%。从231份ELISA检测阳性血清中随机抽取了20份,采用RT-PCR方法进行检测,结果共检出18份样品为BVDV/MV RT-PCR阳性,阴性血清未扩增出E0基因,RT-PCR方法检测结果与ELISA方法符合率为90%。