PCR／PFLP鉴别伉禽白血病病毒

用PCR方法从禽白血病病毒（avian leukosis virus,ALY）亚群毒株RAV－1、RAV－2感染或未感染的SPF鸡胚成纤维细胞（CEF）分别扩增出1．2kb基因手段。RAV－1囊膜基因可初BglⅡ分为2条相近的600bp片段，RAV－2囊膜基因被BamHI分为2条约600bp片段，对照组基因片段（内源性病毒）不被BglⅡ和BamHI切割。结果显示，ALV囊膜基因PCR／RFLP可用于诊断禽白血病和鉴别不同的AVL毒株。     

用PCR检测鸡大肠杆菌耐热肠毒素基因

用１对合成的特异性核苷酸片段,通过聚合酶链反应（ＰＣＲ）,以鸡致病性大肠菌２０个血清型菌株提取的染色体进行扩增,标准阳性对照（ｐＳＬＭ－４００）及扩增到１６个大小的为１５０ｂｐ的ＤＮＡ阳性片段,其它自株及阳性对照株ｐＥＷＤ２９９未扩增到产物,斑点杂交表明,所扩增的阳性条带与标准耐热肠毒素基因（ＳＴ）杂交后呈强阳性。     

聚合酶链反应检测猪细小病毒的研究

本研究成功地建立了检测猪细小病毒（ＰＰＶ）的聚合酶链反应（ＰＣＲ）技术。以１６个核苷酸引物对首次完成了ＰＰＶＤＮＡ结构蛋白ＶＰ１编码区２２８ｂｐ片段的扩增。同样数目的另一引物对，也成功扩增了Ｖｐ２编码区１５８ｂｐＤＮＡ片段。ＰＰＶＢＭ－１株与其它国内分离株（广西株、天津株和哈尔滨株）均出现相同扩增结果。琼脂糖凝胶电泳显示了特异条带（ＶＰ１２２８ｂｐ．Ｖｐ２１５８ｂｐ），而伪狂犬病病毒、犬细小病毒均未扩增，表明了本方法的特异性。同时，限制性内切酶分析（Ｖｐ１２２８ｂｐ及Ｖｐ２１５８ｂｐ分别含单一ＰｖｕⅡ、ＥｃｏＲⅠ位点），也证实了特异性。本方法敏感性高，可检出１０ｆｇ模板ＤＮＡ。既可作样品的快速检测，又能检查细胞系的污染。具有特异、简便、快速的优点。     

鸡痘病毒PCR检测方法的建立

根据已发表的鸡痘病毒 (fowlpox virus,FPV) 4 b核心蛋白基因的核苷酸序列设计合成了 2对引物 ,通过对影响PCR扩增因素的优化 ,2对引物在同一反应条件下 ,以抽提 FPV DNA或直接用其毒液制备的模板均能分别扩增出预期的 5 4 9、136 1bp的片段。特异性检测表明 ,2对引物对 FPV10 2株、FPV2 82 E4 (北京 )、FPV2 82 E4 (吉林 )、v FV2 82重组鸡痘疫苗毒及 FPV临床分离株均能扩增出相应特异性片段 ,而用鸡马立克氏病病毒、鸡传染性喉气管炎病毒、羊痘病毒、鸡胚成纤维细胞 (CEF)制备的模板分别进行 PCR扩增却成阴性。敏感性检测表明 ,2对引物 (p1 ,2 、g1 ,2 )分别能检测到 10 - 2 、10 - 3fmol的 FPV DNA和 10 0 .5、10 - 0 .5TCID50 的病毒。以上结果表明 ,本试验所建立的 FPV PCR检测法具有较高的特异性和敏感性 ,模板制作简单 ,完全可用于 FPV的检测     

肉鸡骨髓细胞瘤病的PCR诊断和病理学研究

对曾感染ALV-J的某肉种鸡场的5只肉鸡进行了PCR检测和病理学研究。结果：5只鸡中有3例PCR呈阳性，5只鸡的骨髓、肝、脾、肾、肺、十二指肠固有膜等组织中均有不同数量的局灶性或单个散在的髓细胞样瘤细胞，瘤细胞胞浆内含有大量圆球形嗜酸性颗粒，表明该鸡场仍有骨髓细胞瘤病的存在。其中2只鸡还同时有增生性肉芽肿，提示有大肠杆菌的混合感染。     

Detection and Identification of Fish-Pathogenic Aphanomyces piscicida Using Polymerase Chain Reaction (PCR) with Species-Specific Primers

Abstract

Aphanomyces piscicida is an important pathogen that plays a role in the morbidity and mortality of various fish species around the world. The poor quality of current identification techniques for this fungus led to the development of highly sensitive and specific polymerase chain reaction (PCR) techniques. Primers derived from the ITS1 and ITS2 regions of A. piscicida NJM 0204 were designed for the detection and identification of fish-pathogenic A. piscicida, with the potential for the diagnosis of mycotic granulomatosis (MG). Polymerase chain reaction amplification showed that the primer set was specific only to fish-pathogenic A. piscicida, not to non-fish-pathogenic Aphanomyces, Saprolegnia spp., Achlya spp., Dictyuchus spp., and Lagenidium spp. In addition, PCR revealed an improved sensitivity sufficient to detect A. piscicida in artificially infected goldfish Carassius auratus. Results demonstrated that the PCR method established in this study is effective for the detection and identification of A. piscicida with MG.     

A Preliminary Trial Using Multi-target Polymerase Chain Reaction (multiplex PCR) and Restriction Fragment Length Polymorphism (PCR-RFLP) on the Same Feedstuffs to Detect Tissues of Animal Origin

A preliminary study using multi-target polymerase chain reaction (multiplex PCR) and restriction fragment length polymorphism (PCR-RFLP) was done on the same feedstuffs to detect animal tissues. The results of the two methods differ somewhat: PCR-RFLP did not detect any signal in any sample, but multiplex PCR detected a signal in one sample. These findings could be a basis for further investigations.     

Quantitative Polymerase Chain Reaction (PCR) for Detection of Aquatic Animal Pathogens in a Diagnostic Laboratory Setting

Abstract

Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.

Received March 30, 2011; accepted May 6, 2011     

用PCR检测副溶血性弧菌耐热溶血素基因

根据副溶血性弧菌的耐热溶血素基因（ｔｄｈ）设计了２对引物，经聚合酶链式反应（ＰＣＲ）检测，所有１８株神奈川试验阳性副溶血性弧菌均呈阳性反应，而６株神奈川试验阴性副溶血性弧菌呈阴性反应，其他９种（３４株）弧菌和８株其他革兰氏阴性菌也呈阴性反应。检测敏感性可达２ＣＦＵ的副溶血性弧菌。     

随机扩增多态DNA（RAPD）技术在鹅育种上的应用

本文用4种引物（OPH5、OPH13、OPH16和OPF4）对隆昌鹅、太湖鹅和新太湖鹅进行基因组DNA、RAPD分析。结果表明：三种鹅都有特异性的条带,扩增DNA条带都表现为多态性,条带数为3-12条,大小范围在0.36-0.38kb之间,多态频率为72.14%;RAPD标记可作为一种辅助手段来比较鹅群体的遗传变异性,太湖鹅遗传变异性大于隆昌鹅;OPF-04很可能用作区分隆昌鹅与太湖鹅的标记性引物。     

Field Evaluation of Sensitivity and Specificity of a Polymerase Chain Reaction (PCR) for Detection of Nucleospora salmonis in Rainbow Trout

Abstract

We determined the sensitivity and specificity of a nested polymerase chain reaction (PCR) for detection of the microsporidian parasite Nucleospora salmonis in kidney tissue of rainbow trout Oncorhynchus mykiss. Kidney tissues were sampled on three dates from 162 juvenile rainbow trout obtained from a California State fish hatchery where the organism was endemic. Kidney tissues were used to prepare imprints stained with May–Grünwald Giemsa and for extraction of genomic DNA for a nested PCR test for N. salmonis. Positive PCR results for N. salmonis were obtained from 1 of 100, 2 of 32, and 27 of 30 kidneys collected on the first, second, and third sample dates, respectively. Kidney tissues from 3 of 27 trout in the third sample that tested positively by PCR also had microscopic evidence of parasites in stained kidney imprints. No parasites were detected in the remaining 159 kidney samples examined microscopically. Sensitivity and specificity of the PCR assay were estimated by using maximum likelihood estimation based on cross-classified test results. This method yielded estimates of sensitivity of 99.99% and specificity of 99.87%. This field evaluation supports experimental evidence that the nested PCR test will be a valuable diagnostic tool for prevention and control of N. salmonis as well as for risk assessment associated with fish movements.     

Genotyping of Mycoplasma gallisepticum and M. synoviae by Amplified Fragment Length Polymorphism (AFLP) analysis and digitalized Random Amplified Polymorphic DNA (RAPD) analysis

Mycoplasma gallisepticum (MG) and M. synoviae (MS) are the cause of considerable economic losses in the poultry industry. Molecular differentiation of avian Mycoplasma strains may be helpful in tracing infections and in the evaluation of implemented intervention strategies. Amplified Fragment Length Polymorphism (AFLP) has shown to be a powerful typing technique but the application for poultry Mycoplasma strains is very limited. The aim of this study was to evaluate the reproducibility and discriminatory power of AFLP HindIII/HhaI and AFLP BglII/Mfel for the inter- and intraspecies differentiation of avian mycoplasmas and to compare these test characteristics with digitalized Random Amplified Polymorphic DNA (RAPD) analysis. The reproducibility of RAPD, AFLP HindIII/HhaI and AFLP BglII/Mfel was 50-100, 97-98 and 86-99%, respectively. RAPD and both AFLP enzyme combinations were able to differentiate between five avian Mycoplasma species. For AFLP, five MG and four MS clusters could be identified. The phylogenetic tree for both enzyme combinations was comparable. For RAPD, four MG clusters could be identified. For MS, however, due to the poor reproducibility of the RAPD technique, no clear genogroups could be identified. On basis of the results of this study it can be concluded that AFLP is a powerful technique for the genotyping of avian mycoplasmas and that, although AFLP HindIII/HhaI generated patterns with less fragments, the final results showed homologous results.     

Failure of the Polymerase Chain Reaction (PCR) Method to Detect Striped Jack Nervous Necrosis Virus (SJNNV) in Striped Jack Pseudocaranx dentex Selected as Spawners

Abstract

The causative agent of viral nervous necrosis (VNN) in striped jack Pseudocaranx dentex is transmitted from female and male spawners to larvae, and elimination of carrier spawners, determined by the detection of striped jack nervous necrosis virus (SJNNV) via polymerase chain reaction (PCR), has been used to prevent transmission of the disease in larval production facilities. However, some outbreaks of VNN occurred in larvae obtained from SJNNV-negative spawners. We compared the occurrence of infection between groups of larvae obtained from SJNNV-negative spawners and those from SJNNV-positive spawners. Viral nervous necrosis occurred in all seven groups of larvae obtained from the virus-positive spawners between the 3rd and 7th day of rearing. The virus was detected only occasionally after 2 weeks in four of six groups obtained from the virus-negative spawners. These results confirmed vertical transmission of the virus and revealed that a very small amount of SJNNV in spawners escaped PCR detection and could produce infection, although the frequency of infection occurring in the SJNNV-negative group was much lower than that among the larvae from SJNNV-positive spawners.     

Determination of Phylogenetic Relationships of Flavobacterium psychrophilum (Flexibacter psychrophilus), Flavobacterium columnare (Flexibacter columnaris), and Flexibacter maritimus by Sequence Analysis of 16S Ribosomal RNA Genes Amplified by Polymerase Chain Reaction

Abstract

The nearly complete small subunit 16S ribosomal RNA (rRNA) gene sequence amplified by polymerase chain reaction was determined for Flavobacterium psychrophilum (formerly Flexibacter psychrophilus) by using automated nucleotide sequencing. The sequence was found to be 1,465 base pairs (bp) in length, a size consistent with previously determined sequences for 14 other bacterial species from various taxa, including the yellow-pigmented bacteria. Sequence signatures confirmed that this organism was a member of the bacterial division Bacteroides–Flavobacterium. Parsimonious and additive phylogenetic trees were constructed with homology, and pairwise evolutionary distances were used to estimate phylogenetic relationships. Data show that F. psychrophilum, F. columnare, and Flexibacter maritimus are closely related, have a common descent, and represent a distinct group within the division Bacteroides–Flavobacterium. This group also included other organisms from the genera Flavobacterium and Cytophaga. Further, Flavobacterium aquatile, the type species for the genus Flavobacterium, was also determined to be a member of this “Flavobacterium–Cytophaga–Flexibacter subcomplex.” This supports previous assertions that the type strain of Flavobacterium should be changed to a more representative species such as Flavobacterium breve.     

Polymerase chain reaction (PCR) amplification of parvoviral DNA from the brains of dogs and cats with cerebellar hypoplasia

Cerebellar hypoplasia in cats is caused most commonly by an in utero or perinatal infection with feline panleukopenia virus (parvovirus). Cerebellar hypoplasia has been reported infrequently in dogs, but no viral etiology has been identified to date. DNA was extracted from archival, paraffin-embedded, cerebellar tissue from 8 cats and from 2 canine littermates with cerebellar hypoplasia, 2 canine littermates with cerebellar cortical abiotrophy, 6 dogs with congenital cerebellar vermal defects, 1 dog with congenital hydranencephaly, and 15 dogs and cats with various encephalitdes. The DNA extracted from each cerebellum was subject to polymerase chain reaction (PCR) amplification by 3 primer pairs specific for parvovirus DNA. Sequence analysis of PCR products from each of the 8 cats and 2 dogs with cerebellar hypoplasia confirmed their identity with parvoviral DNA. The 6 dogs with cerebellar vermal defects, 2 dogs with cortical abiotrophy, 1 dog with congenital hydranencephaly, and all control samples were PCR negative for parvovirus. Parvoviral structural proteins were not identified by immunohistochemistry in either dog with cerebellar hypoplasia. This study shows that parvoviral DNA can be amplified from feline and canine archival brain tissue and that cerebellar hypoplasia in dogs might be associated with in utero parvovirus infection.     

用RAPD技术检测不同来源微孢子虫基因组DNA多态性

家蚕微粒子病是由微粒子原虫（微孢子虫）类寄生于蚕体引起的病害。微粒子病对蚕丝业生产危害极大,一直被列为蚕种制造检疫对象。许多学者对病原的传播途径、侵染机制及分类地位作了大量研究。其中微孢子虫的分类又以血清学关系、发育增殖方式和孢子超微结构作为主要划分依据,这些表型依据仍然具有局限性。随机扩增多态性ＤＮＡ技术（ＲａｎｄｏｍｌｙＡｍｐｌｉｆｉｅｄＰｏｌｙｍｏｒｐｈｉｃＤＮＡ,ＲＡＰＤ）因能方便、快速提供ＤＮＡ多态性的丰富信息而被广泛用于动植物和微生物的遗传差异、亲缘关系和进化分类等领域,但应用于微…     

A Quantitative Polymerase Chain Reaction Assay for the Detection and Quantification of Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus 3)

Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R² = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR.

Received April 20, 2015; accepted November 10, 2015     

应用聚合酶链式反应检测禽痘病毒田间分离株及疫苗株中网状内皮组织增殖病毒LTR片断,env及re1基因

本文利用聚合酶链式反应对来自澳大利亚昆士兰州、维多利亚州和新南维尔士州的４株禽痘病毒田间分离株及二疫苗株中网状内皮组织增殖病毒的ＬＴＲ片断、ｅｎｖ及ｒｅｌ基因因进行了检测。所有４株痘病毒分离株均为ＬＴＲ片断、ｅｎｖ阳性,曾被ＰＣＲ证实为ＬＴＲ阳性的一疫苗株在本实验中也为ＬＴＲ、ｅｎｖ阳性,另一疫苗株为ＬＴＲ、ｅｎｖ阴性,所有上述禽痘病毒株均为ｒｅｌ阴性。