DNA Quantity and Quality in Remnants of Traffic-Killed Specimens of an Endangered Longhorn Beetle: A Comparison of Different Methods

The sampling of living insects should be avoided in highly endangered species when the sampling would further increase the risk of population extinction. Nonlethal sampling (wing clips or leg removals) can be an alternative to obtain DNA of individuals for population genetic studies. However, nonlethal sampling may not be possible for all insect species. We examined whether remnants of traffic-killed specimens of the endangered and protected flighless longhorn beetle Iberodorcadion fuliginator (L., 1758) can be used as a resource for population genetic analyses. Using insect fragments of traffic-killed specimens collected over 15 yr, we determined the most efficient DNA extraction method in relation to the state of the specimens (crushed, fragment, or intact), preservation (dried, airtight, or in ethanol), storage duration, and weight of the sample by assessing the quantity and quality of genomic DNA. A modified cetyltrimethyl ammonium bromide method provided the highest recovery rate of genomic DNA and the largest yield and highest quality of DNA. We further used traffic-killed specimens to evaluate two DNA amplification techniques (quantitative polymerase chain reaction [qPCR] and microsatellites). Both qPCR and microsatellites revealed successful DNA amplification in all degraded specimens or beetle fragments examined. However, relative qPCR concentration and peak height of microsatellites were affected by the state of specimen and storage duration but not by specimen weight. Our investigation demonstrates that degraded remnants of traffic-killed beetle specimens can serve as a source of high-quality genomic DNA, which allows to address conservation genetic issues.     

Development and application of molecular methods (PCR) for detection of Tasmanian Atlantic salmon reovirus

Molecular (PCR) diagnostic tests for the detection and identification of aquareovirus in general, and Tasmanian Atlantic salmon reovirus (TSRV) specifically, were developed, and their diagnostic sensitivity and specificity were determined and compared with virus isolation in cell culture. Intralaboratory and interlaboratory comparison of PCR (conventional hemi‐nested RT‐PCR & RT‐qPCR) and virus isolation in cell culture using finfish cell lines, CHSE‐214 and EPC, was carried out for the detection and identification of TSRV using field samples of farmed Atlantic salmon Salmo salar, L. from various aquaculture sites around Tasmania. The interlaboratory comparison of diagnostic methods was carried out between two laboratories, AAHL‐CSIRO and DPIPWE‐Tasmania. A total of 144 fish from nine sites (12–33 fish per site) were sampled from two regions of Tasmania (Tamar River estuary in the north and Huon River estuary in the south‐east) during late spring to early summer of 2009, and the data were analysed using different statistical approaches. The prevalence of TSRV ranged from 6% to 22% in both regions. All the diagnostic methods (data from both laboratories) had high specificity, while the estimated sensitivity varied between tests with RT‐qPCR being the most sensitive (95.2%) method followed by virus isolation and then conventional hemi‐nested RT‐PCR.     

应用纳米磁珠实时荧光定量PCR方法检测番茄黄化曲叶病毒

In order to develop a rapid, sensitive and specific qPCR assay for detection and quantification of Tomato yellow leaf curl virus (TYLCV), a pair of primers and TaqMan probe were designed according to the conserved sequence of known TYLCV isolates. Combining with MNP technique, a novel MNP-qPCR detection method was established and verified based on specificity, sensitivity and reproducibility tests. The results indicated that the Ct value of plotted standard curve showed good linear relationship(R² =0.9994)with the log of copy number of template. The established method showed a high specificity for TYLCV detection without crossing reaction with Tomato severe leaf curl virus and Tomato yellow leaf curl Sadinia virus, and was 10-fold more sensitive than routine PCR. Both coefficients of variation were less than 2%, indicating a good reproducibility. We have provided a novel method for detection of TYLCV in plant samples rapidly and quantitatively.     

High HEV presence in four different wild boar populations in East and West Germany

Swine Hepatitis E virus (HEV) can be transmitted from pigs to humans causing hepatitis. A high prevalence of HEV in wild boar populations is reported for several European countries, but actual data for Germany are missing. During the hunting season from October to December 2007 liver, bile and blood samples were collected from wild boars in four different German regions. The samples were tested for HEV RNA by quantitative PCR (qPCR) and anti-HEV IgG antibodies by two different ELISAs and a Line immunoassay. A seroprevalence of 29.9% using ELISA and 26.2% in the Line immunoassay was determined. The seroprevalence rate varied greatly within the analyzed regions. However, qPCR analysis revealed a higher prevalence of 68.2% positive animals with regional differences. Surprisingly, also adult wild sows and wild boars were highly HEV positive by qPCR. Compared to liver and serum samples, bile samples showed a higher rate of positive qPCR results. Sequencing and phylogenetic analysis of a 969 nt fragment within ORF 2 revealed that all isolates clustered within genotype 3 but differed in the subtype depending on the hunting spot. Isolates clustered within genotypes 3i, 3h, 3f and 3e. Within one population HEV isolates were closely related, but social groups of animals in close proximity might be infected with different subtypes. Two full-length genomes of subtypes 3i and 3e from two different geographic regions were generated. The wild boar is discussed as one of the main sources of human autochthonous infections in Germany.     

Development and validation of a range of endogenous controls to support the implementation of practical Taqman real-time PCR-based surveillance for fish diseases within aquaculture

The use of Taqman real-time PCR-based technology has recently become more frequent in the detection of pathogens in the aquaculture industry. This interest has necessitated the development of robust and reliable pathogen-detection assays. The development of a range of endogenous control assays to be run alongside these diagnostic assays works to further increase confidence in the latter. This study describes the design of a range of endogenous control assays based on the elongation factor 1-α (EF1-α) gene specific to a range of fish species including Atlantic salmon, Salmo salar; rainbow trout, Oncorhynchus mykiss; brown trout, Salmo trutta; cod, Gadus morhua; haddock, Melanogrammus aeglefinus; saithe, Pollachius virens; whiting, Merlangius merlangus; Norway pout, Trisopterus esmarkii; carp (family Cyprinidae), roach, Rutilus rutilus; European eel, Anguilla anguilla; and herring, Clupea harengus, as well as a number of fish cell lines. Evidence is provided of the validation of these assays for specific species, a range of tissue types and cell lines as well as an example of the potential uses of these assays.     

基于荧光定量PCR的鳜传染性脾肾坏死病毒滴度检测方法

针对传染性脾肾坏死病毒(ISKNV)的ORF007基因,设计特异性引物及TaqMan探针,建立了ISKNV的实时荧光定量PCR方法。采用CPE法对连续10倍稀释的ISKNV培养液的滴度进行了测定,同时采用荧光定量PCR法测定病毒拷贝数。结果显示,荧光定量PCR测定的病毒拷贝数与CPE法测定的病毒滴度具有良好的线性关系,其线性方程为y=1.076x+0.545(R2=0.998 6),其中y为基因组拷贝数浓度的对数,x为病毒滴度TCID50的对数。研究表明,荧光定量PCR法可替代CPE法应用于ISKNV疫苗抗原的定量,大大缩短了疫苗制备的时间,为疫苗生产提供了方便。     

香猪睾丸miR-449b和miR-222分子的发育性变化

为了阐明miR-449b和miR-222对贵州从江香猪睾丸发育的调节作用。运用qPCR方法检测香猪睾丸组织中miR-449b和miR-222表达量的发育性变化,通过miRSystem数据库、Kobas数据库对预测的靶基因进行信号转导通路富集分析。经克隆测序,得到香猪miR-222序列,与猪的相似性为100%,而且物种之间miR-449b和miR-222两个分子的保守性都很高。香猪睾丸中miR-449b于1~4月龄期间的表达量逐渐上升,4月龄时表达量达到峰值,之后下降;miR-222的表达量呈现相反的变化趋势,于1~4月龄期间表达量逐渐下降,1月龄时最高,4月龄时最低,之后的表达量有所回升;已知物种的miR-449b和miR-222序列高度保守;经预测miR-449b靶基因有420个,富集于卵母细胞减数分裂、Notch信号通路、Ras信号通路等100个途径(P0.05),miR-222的靶基因有429个,富集于细胞周期、Fox O信号通路、TGF-β信号通路等69个通路(P0.05);miR-449b和miR-222的靶基因共享的通路为Wnt信号通路、MAPK信号通路、雌激素信号通路等42个(P0.05)。     

Occurrence of canine hemotropic mycoplasmas in domestic dogs from urban and rural areas of the Valdivia Province,southern Chile

This is the first study to investigate the occurrence, risk factors and hematological findings of hemoplasmas in dogs from Chile. Complete blood count and 16S rRNA conventional PCR for Mycoplasma spp. were performed in 278 blood samples from rural (n = 139) and urban (n = 139) dogs in Valdivia. Real time 16S rRNA PCR (qPCR) allowed species identification. Mycoplasma spp. occurrence was 24.8%. ‘Candidatus M. haematoparvum’ (CMhp) was identified in 12.2% and Mycoplasma haemocanis (Mhc) in 11.9% dogs. It was not possible to identify species in two Mycoplasma spp. samples by qPCR. Sequencing allowed identifying one of them as ‘Candidatus M. turicensis’ (CMt). Frequency in rural localities was higher (41.7%) than in urban (7.9%). Rural locality, maleness and older age were risk factors for hemoplasmosis. Hemoplasma-positive dogs had a higher total protein. This is the first report of Mhc, CMhp and CMt in dogs from Chile, with a high occurrence in rural localities.     

鸭疫里默氏杆菌实时荧光定量PCR快速检测方法的建立与应用研究

根据GenBank登录的鸭疫里默氏杆菌外膜蛋白(OmpA)基因保守区序列,设计引物和探针,建立了直接检测鸭疫里默氏杆菌的实时荧光定量PCR快速方法。10倍梯度稀释RA菌株制备DNA模板测定方法的灵敏度,结果可在8.0×103CFU/mL浓度下特异地检测出目的菌,最低能检测到RA的DNA模板为8.0拷贝/μL;对鸭源大肠杆菌、多杀性巴氏杆菌、沙门氏菌、变形杆菌、金黄色葡萄球菌进行检测,结果均为阴性,建立的实时荧光定时PCR方法特异性强。用RA菌株人工感染雏鸭,感染后定时采集咽喉拭子、泄殖腔拭子、心血、肝脏、肺脏、脑,分别用实时荧光定量PCR检测,对脏器进行病原菌分离。结果表明,1/10半数致死量接种组,感染后8 h从心血、肝脏、脑组织检测到RA核酸,16 h后全部试样呈阳性;24 h首先从肝脏分离到RA,心血、肝脏、肺脏、脑组织均分离出RA。心血和脏器RA实时荧光定量PCR阳性检出率为63.8%(51/80),RA分离率为28.8%(23/80)。10个半数致死量接种组,1 h即可从咽喉拭子、心血和肝检测到RA核酸,5 h全部样品为阳性;5 h从肺和脑分离到RA。RA人工感染鸭的心血、肝脏、肺脏、脑实时荧光定量PCR阳性检出率为86.3%(69/80),RA分离率为60%(48/80)。用加热裂解法提取样品RA DNA只需30 min,建立的实时荧光定量PCR检测RA只需2 h,适用于RA的快速诊断、流行病学调查、种鸭引进隔离检疫、活鸭及其产品国内市场和进出口检验检疫。