牛蒡上发生的根结线虫种类鉴定

2007～2009年,对我国牛蒡主产区的牛蒡及其根际土壤中的植物寄生线虫进行调查研究,发现根结线虫危害最为严重。根据根结线虫的形态学特征和分子生物学检测方法对其进行鉴定,结果表明,我国牛蒡主产区根结线虫种类为北方根结线虫(Meloidogyne hapla)。     

番茄溃疡病菌PCR快速检测技术

番茄溃疡病是一种严重危害番茄生产的细菌性病害，许多国家将其列为检疫性病害。利用ITS通用引物扩增了番茄溃疡病菌（Clavibacter michiganensis subsp．michiganensis）的ITS序列，并进行克隆测序。根据序列比较结果设计了引物BT1和BT2，该引物特异性好，能专一扩增出268bp电泳条带，而马铃薯环腐病菌等不同亚种、不同属的细菌及健康的番茄材料均无扩增条带。从接种但未显症番茄苗叶片及人工模拟染菌种子上提取总DNA，以此为模板均能稳定地扩增出特异性目的条带。该方法直接对种子或植株进行检测，不需进行病原菌分离培养，快速简便，适用于出入境检验检疫及种苗健康检测领域。     

双重PCR检测马铃薯晚疫病和环腐病方法的建立

通过克隆马铃薯环腐病菌和晚疫病菌转录间隔区(ITS)序列,并对测序结果进行同源性比较,选取差异位点分别设计了两对引物P.IN1/P.IN2和C.IN1/C.IN2,并检测了引物的特异性及方法的灵敏度。引物P.IN1/P.IN2可扩增出1条363bp马铃薯晚疫病菌的特异性条带,在DNA水平上其灵敏度达18fg/μL;引物C.IN1/C.IN2可扩增出1条218bp马铃薯环腐病菌的特异性条带,在细菌数上检测灵敏度为104 cfu/mL。混合这两对引物构建双重PCR反应体系,能从马铃薯环腐病菌和晚疫病菌的混合DNA及感染这两种菌的马铃薯植株中同时扩增到363bp和218bp的特异片段。实现了同时对马铃薯晚疫病菌和环腐病菌的快速可靠检测。     

Studies on amoebae and cysts associated with the isolation of Spongospora subterranea f.sp. subterranea in vitro

New evidence is presented to support the contention that the amoeba/cyst colonies isolated from surface-sterilized Spongospora subterranea f.sp. subterranea -infected potato tubers and spore balls have a saprophytic phase but are contaminants and not S. subterranea. Amoebae isolated from infected tissues and spore balls formed colonies associated with bacteria on 1% water agar at 18°C and encysted after 5–7 days. These cysts were morphologically distinct from the resting spores of S. subterranea and were formed singly or in a layer, unlike the spore ball (cystosorus) of S. subterranea . Amoebae, cysts and mixtures of amoebae and cysts in primary, secondary and tertiary subcultures failed to infect tomato roots. PCR amplification of DNA from amoebae, cysts and spore balls using the S. subterranea -specific primer pair SsF/R generated a 434-bp product from S. subterranea spore balls only and not from amoebae or cysts. When an amoeba/cyst-specific primer pair AmF/R was designed and used for PCR amplification, a single 411-bp product was generated from DNA of amoebae and cysts, but not from DNA of S. subterranea spore balls. These results are discussed in relation to earlier reports claiming the successful isolation of S. subterranea and other plasmodiophorids in vitro .     

采用通用引物PCR配合SSCP和RFLP技术检测鱼病病原菌

采用通用引物PCR（UPPCR）、PCR－RFLP、PCR－SSCP技术,研究快速鉴别鱼病病原菌的分子生物学诊断技术。结果发现,采用细菌16S rRNA基因保守区特异性引物,以嗜水气单胞菌、鲁克氏耶尔森菌、鳗弧菌、柱状曲挠杆菌、乙型链球菌、荧光假单胞菌等部分常见鱼病病原菌为对象,可以建立一种UPPCR技术。该技术能在保证实验条件不变的基础上,检出上述所有细菌,并还可检出大肠杆菌和双歧杆菌等非鱼病病原菌。并且认为,该法与SSCP配合即采用UPPCR－SSCP技术能较好地鉴别被检菌而用于鱼病病原菌的快速诊断。     

鸡基因组DNA不同提取方法的比较研究

实验用优化煮沸法提取鸡血液基因组DNA,经核酸蛋白定量仪检测,DNA的OD260/OD280达1.77±0.06,证明所提DNA质量较好,用于PCR扩增,可以得到满意的扩增效果。与酚-氯仿法相比,优化煮沸法避免了用氯仿、酚等剧毒试剂,且节约了时间和成本,是一种较好的提取基因组DNA的方法。     

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。     

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.     

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。     

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。