Use of nested polymerase chain reaction (PCR) for detection of retroviruses from formalin-fixed, paraffin-embedded uveal melanomas in cats

Thirty-six formalin-fixed, paraffin-embedded enucleated globes from cats with a diagnosis of diffuse anterior uveal melanoma were obtained. Sections of tumor were excised, deparaffinized, and subjected to nested polymerase chain reaction (PCR) to identify proviral DNA sequences from the feline leukemia virus (FeLV)–feline sarcoma virus (FeSV; 36 eyes), and the feline immunodeficiency virus (FIV; 18 eyes). All samples tested were negative for FIV DNA. Three samples were positive for FeLV–FeSV DNA. This is the first reported evidence of a possible link between naturally occurring feline anterior uveal melanoma and the presence of FeLV–FeSV DNA.     

Characterisation of lymphosarcomas in Australian cats using polymerase chain reaction and immunohistochemical examination

Objective To examine tumour tissue of cats with lymphosarcoma for the presence of feline leukaemia virus and feline immunodeficiency virus and analyse the immunophenotype of the tumours.
Design A retrospective study of feline lymphosarcoma cases.
Methods Formalin-fixed, paraffin-embedded tumour tissue of 14 feline lymphosarcomas was examined for the presence of feline leukaemia virus and feline immunodeficiency virus by polymerase chain reaction and immunohistochemistry. Using polyclonal and monoclonal antibodies against T and B lymphocytes, the phenotypic expression of the tumours was characterised.
Results No feline leukaemia virus antigen or proviral sequences were detected. Feline immunodeficiency virus proviral sequences were detected in two cases by polymerase chain reaction. Immunophenotyping of all 14 cases resulted in seven cases being classified as B-cell phenotype, four as T-cell phenotype, and the remaining three undetermined.
Conclusions In contrast to previous reports overseas, our results suggest that feline leukaemia virus infection appears to be an infrequent cause of lymphosarcoma in the cats that were necropsied. Feline immunodeficiency virus may have a role in lymphomagenesis. The potential role of feline immunodeficiency virus needs to be explored in more depth. Compared with most previous reports, B-cell tumours were more common than T-cell tumours in this series of cats.     

Development and clinical evaluation of a rapid diagnostic kit for feline leukemia virus infection

Feline leukemia virus (FeLV) causes a range of neoplastic and degenerative diseases in cats. To obtain a more sensitive and convenient diagnosis of the disease, we prepared monoclonal antibodies specific for the FeLV p27 to develop a rapid diagnostic test with enhanced sensitivity and specificity. Among these antibodies, we identified two clones (hybridomas 8F8B5 and 8G7D1) that specifically bound to FeLV and were very suitable for a diagnostic kit. The affinity constants for 8F8B5 and 8G7D1 were 0.35 × 10⁹ and 0.86 × 10⁹, respectively. To investigate the diagnostic abilities of the rapid kit using these antibodies, we performed several clinical studies. Assessment of analytical sensitivity revealed that the detection threshold of the rapid diagnostic test was 2 ng/mL for recombinant p27 and 12.5 × 10⁴ IU/mL for FeLV. When evaluating 252 cat sera samples, the kit was found to have a kappa value of 0.88 compared to polymerase chain reaction (PCR), indicating a significant correlation between data from the rapid diagnostic test and PCR. Sensitivity and specificity of the kit were 95.2% (20/21) and 98.5% (257/261), respectively. Our results demonstrated that the rapid diagnostic test would be a suitable diagnostic tool for the rapid detection of FeLV infection in cats.     

Evaluation of barcoded magnetic bead–based immunoassays for the simultaneous detection of feline leukemia virus antigen and antibody against feline immunodeficiency virus

Testing platforms that leverage automation, require minimal sample volume, and enable various tests to be performed simultaneously on a single sample have the potential to improve workflow and efficiency in veterinary diagnostic laboratories. We evaluated a barcoded magnetic bead (BMB) technology using established immunoassays for detection of feline leukemia virus (FeLV) p27 antigen and antibody against feline immunodeficiency virus (FIV). Analytical sensitivity, limit of blank, and limit of detection were used to establish a functional sensitivity of 1.00 ng/mL of inactivated FeLV antigen and 35.7 ng/mL of anti-FIV monoclonal antibody. Common interferents, such as hemoglobin, lipid, and bilirubin, were not found to interfere with the performance of the assay. Intra- and inter-assay CVs were <13% for both assays using manufactured samples. Using a set of 116 feline samples, the diagnostic accuracy of our multiplex assay was 100% compared to reference assays. Performance in a convenience set of 1,000 feline samples submitted to a commercial diagnostic laboratory revealed a proportion of positive results of 1.3% for FeLV and 3.7% for FIV. BMB technology should enable rapid screening of samples for various markers in a single immunoassay well.     

检测和鉴别犬瘟热病毒强、弱毒株的复合反转录-套式聚合酶链式反应(RT-nPCR)的建立及初步应用

根据GenBank上登录的犬瘟热病毒（Canine distemper virus，CDV）基因组全序列，选择CDV强、弱毒株间有区别保守区设计了一对通用引物P1和P4，并在该对引物跨越区域的内部设计了CDV强毒株特异性引物P2及弱毒株特异性引物P3，用引物P1／P4进行RT—PCR，然后用引物P2／P3／P4进行复合套式PCR，建立了一种能区分CDV强、弱毒株的复合反转录-套式聚合酶链式反应（RT—nPCR）的鉴别诊断方法。应用该方法从CDV强、弱毒株的基因组中分别扩增出了大小为247bp和177bp的特异性片段，从两种病毒基因组混合物中扩增出了大小为247bp和177bp的两条特异性片段，与犬细小病毒、犬腺病毒、犬冠状病毒、狂犬病病毒、新城疫病毒的细胞培养物以及正常细胞对照组进行复合RT—nPCR扩增时均为阴性。对从黑龙江省和吉林省采集的20份疑似CDV病料进行的检测结果表明，有15份类似CDV强毒，5份类似CDV弱毒。本研究建立的复合RT—nPCR可以有效检测CDV感染，能够将强、弱毒株区分开，可用于临床快速检测、流行病学监测以及追踪疫苗免疫效果等。     

Application of the polymerase chain reaction (PCR) in veterinary diagnostic virology

The polymerase chain reaction has become an important diagnostic tool for the veterinary virologist. Conventional methods for detecting viral diseases can be laborious or ineffective. In many cases PCR can provide a rapid and accurate test. In this article we explain the basic principles of PCR and supply a reference list of its uses in diagnostic veterinary virology.Abbreviations BLV bovine leukaemia virus - BVDV bovine viral diarrhoea virus - DNA deoxyribonucleic acid - ds double-stranded - ELISA enzyme-linked immunosorbent assay - PCR polymerase chain reaction - RNA ribonucleic acid - ss single-stranded - TK thymidine kinase     

Lack of detection of feline leukemia and feline sarcoma viruses in diffuse iris melanomas of cats by immunohistochemistry and polymerase chain reaction.

Diffuse iris melanoma was confirmed by light-microscopic examination in 10 formalin-fixed, paraffin-embedded globes from 10 cats. To determine if feline leukemia virus or a replication defective feline leukemia virus, feline sarcoma virus, was present in these anterior uveal melanomas, immunohistochemistry and polymerase chain reaction for feline leukemia virus were utilized. Immunohistochemical staining for feline leukemia virus glycoprotein 70 was performed on all 10 tumors using an avidin-biotin complex technique. The DNA was extracted from each specimen and a 166-base pair region of the feline leukemia virus long terminal repeat was targeted by polymerase chain reaction. Immunohistochemical staining for feline leukemia virus glycoprotein 70 and polymerase chain reaction amplification of a feline leukemia virus long terminal repeat region were negative in all cases. Feline leukemia virus/feline sarcoma virus was not detected in any neoplasms and therefore was unlikely to play a role in the tumorigenesis of these feline diffuse iris melanomas.     

新型反转录-复合套式聚合酶链式反应(RT-nPCR)鉴别猪瘟强毒和弱毒疫苗方法的建立

对GenBank中登录的25株猪瘟病毒强毒株和兔化弱毒疫苗株基因组全序列进行比较分析,在其高度保守区设计1对通用引物,扩增片段为609bp,并在该对引物扩增区域内设计针对疫苗弱毒的特异引物,扩增片段为237bp,建立一种能够区分猪瘟病毒强毒和疫苗弱毒的敏感、特异、重复性好的反转录-复合套式聚合酶链式反应(RT-nPCR)鉴别诊断方法。该方法能将我国大陆地区流行的不同基因亚群的猪瘟病毒强毒与疫苗弱毒完全区分开来,且不与牛病毒性腹泻病毒及其他猪源病毒发生非特异反应。应用本试验建立的反转录-复合套式聚合酶链式反应(RT-nPCR)方法可以及早对猪瘟作出准确诊断,并可将强毒感染猪迅速从弱毒疫苗免疫猪群中筛选出来,减少了未感染免疫猪被误杀的可能性。     

A micro-enzyme-linked immunosorbent assay (ELISA) test for detection of feline leukemia virus in cats

The performance of a micro ELISA test for detection of feline leukemia virus (FeLV) infection was evaluated. The test was found specific for FeLV and feline sarcoma virus (FeSV) group-specific antigens in blood, plasma or serum of infected cats. Other common feline pathogens were negative to the test.Quantities as little as 7.8 ng of p-27 (the major group specific antigen of FeLV) per ml of sample gave positive results. The correlation between the micro ELISA test and the indirect immunofluorescent test commonly used for diagnosis of FeLV infection was 98% in 116 clinical cases and 184 samples from cats inoculated with FeLV and 100% in 100 specific pathogen-free cats.     

Optimization of polymerase chain reaction for detection of Clostridium botulinum type C and D in bovine samples

Botulism is a rare but serious paralytic illness caused by a nerve toxin that is produced by the bacterium Clostridium botulinum. The economic, medical and alimentary consequences can be catastrophic in case of an epizooty. A polymerase chain reaction (PCR)-based assay was developed for the detection of C. botulinum toxigenic strains type C and D in bovine samples. This assay has proved to be less expensive, faster and simpler to use than the mouse bioassay, the current reference method for diagnosis of C. botulinum toxigenic strains. Three pairs of primers were designed, one for global detection of C. botulinum types C and D (primer pair Y), and two strain-specific pairs specifically designed for types C (primer pair VC) and D (primer pair VD). The PCR amplification conditions were optimized and evaluated on 13 bovine and two duck samples that had been previously tested by the mouse bioassay. In order to assess the impact of sample treatment, both DNA extracted from crude samples and three different enrichment broths (TYG, CMM, CMM followed by TYG) were tested. A 100% sensitivity was observed when samples were enriched for 5 days in CMM followed by 1 day in TYG broth. False-negative results were encountered when C. botulinum was screened for in crude samples. These findings indicate that the current PCR is a reliable method for the detection of C. botulinum toxigenic strains type C and D in bovine samples but only after proper enrichment in CMM and TYG broth.     

Detection of neoplastic lymphocytes in peripheral blood of dogs with lymphoma by polymerase chain reaction for antigen receptor gene rearrangement

BACKGROUND: Uniquely rearranged immunoglobulin and T-cell receptor gene sequences can be amplified and electrophoretically separated by size to detect a clonal population of lymphocytes. OBJECTIVE: The purpose of this study was to determine whether the polymerase chain reaction (PCR) detects neoplastic (clonal) lymphocytes more frequently than do microscopic methods. METHODS: We identified neoplastic lymphocytes in peripheral blood by both routine and standardized microscopic examination of blood smears and by PCR amplification of blood-derived DNA and compared the 3 methods for frequency of detection of leukemic involvement. For standardized microscopic examination (200 leukocytes counted on Wright-Giemsa-stained blood smears), samples were categorized as negative (1% prolymphocytes, no lymphoblasts), or positive (>/=1 lymphoblast). A PCR-amplified sample was positive if 1 or 2 discrete bands were seen on the gel, or negative if no bands, a smear, or a faint ladder was seen. RESULTS: Using PCR, neoplastic lymphocytes were detected in peripheral blood 2.5 times more frequently than with routine or standardized microscopic evaluation. Eighty-three percent of samples negative by microscopy were positive by PCR. CONCLUSION: PCR is more sensitive than microscopy for the detection of clonal lymphocytes in peripheral blood. The results of this study also suggest that neoplastic lymphocytes circulate in peripheral blood at a higher frequency than previously reported. PCR may be useful for detecting or phenotyping lymphoma, monitoring response to therapy, identifying recurrence, and screening breeds at risk.     

Bovine leukemia virus genotype surveillance in cattle at a slaughterhouse in Aichi Prefecture,Japan, in 2019 using polymerase chain reaction combined with restriction fragment length polymorphism

Polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) is commonly used for genotyping bovine leukemia virus (BLV) in slaughterhouses. However, unclassified BLV genotypes have been sporadically reported. To assess the current status of BLV genetic characterization in cattle, PCR-RFLP was performed on blood samples of 170 cattle (84 Japanese Black, 60 Japanese Black x Holstein, and 26 Holstein) from 17 farms (5 prefectures) at a slaughterhouse in Aichi Prefecture in 2019. A total of 65 samples (38.2%) were BLV positive, and genotype 1 was the most predominant (56/65 samples), followed by genotypes 3 (6 samples) and 5 (1 sample), and two unclassified samples. No relationship between the genotypes and breeds was observed. Sequence and phylogenetic analyses demonstrated that unclassified BLV genotypes clustered with genotype 1 sequences were, therefore, not new genotypes.     

PCR技术检测饲料中沙门氏菌的应用研究

用聚合酶链反应(PCR)技术检测饲料中沙门氏菌,对已知被沙门氏菌污染的动物饲料培养物均能检出阳性,说明本方法对检测饲料中沙门氏菌具有特异性高、灵敏、快速等特点,适用于快速、准确地检测饲料中沙门氏菌的需要。     

Identification of equine herpesviruses 1 and 4 by polymerase chain reaction

OBJECTIVE: To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus). DESIGN: Primer sets based on nucleotide sequences encoding glycoprotein H (gH) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections. METHODS: Oligonucleotide primers were designed for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The tests were applied to tissue samples from aborted equine foetuses and to nasopharyngeal swabs from horses with acute febrile respiratory disease. RESULTS: Individual single round and a second round (seminested) EHV1 and EHV4 PCRs were specific in that EHV1 primers amplified all (n = 30) EHV1 isolates and did not amplify EHV4. Similarly EHV4 primers amplified all (n = 6) EHV4 isolates and did not amplify EHV1. Both PCRs were sensitive in that the first round EHV1 PCR detected 1220 molecules of EHV1 plasmid DNA and the first round EHV4 PCR detected 7280 molecules of EHV4 plasmid DNA. The EHV1 second round PCR was 100 times more sensitive in that it detected 12 molecules of EHV1 DNA and the EHV4 second round PCR was 1000 times more sensitive in that it detected 8 molecules of EHV4 DNA. There was a high correlation between detection of EHV1 by virus isolation and PCR when tissue samples from 71 aborted foetuses were examined; all samples positive by virus isolation were positive by PCR. Similarly the EHV4 PCR was at least as sensitive as virus isolation when applied to nasaopharyngeal swabs from horses with respiratory disease in that all samples positive by virus isolation were also positive by PCR. CONCLUSION: Individual single round and second round (seminested) PCRs and a seminested multiplex PCR were developed that enabled reliable, rapid detection of EHV1 and EHV4 in aborted foetal tissues and nasopharyngeal swab samples.     

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。     

A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen

A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reaction. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.Abbreviations AI artificial insemination - BHV1 bovine herpesvirus type 1 - PCR polymerase chain reaction - TCID₅₀ tissue culture infective dose, 50%     

Real-time polymerase chain reaction: a novel molecular diagnostic tool for equine infectious diseases

The focus of rapid diagnosis of infectious disease of horses in the last decade has shifted from the conventional laboratory techniques of antigen detection, microscopy, and culture to molecular diagnosis of infectious agents. Equine practitioners must be able to interpret the use, limitations, and results of molecular diagnostic techniques, as they are increasingly integrated into routine microbiology laboratory protocols. Polymerase chain reaction (PCR) is the best-known and most successfully implemented diagnostic molecular technology to date. It can detect slow-growing, difficult-to-cultivate, or uncultivatable microorganisms and can be used in situations in which clinical microbiology diagnostic procedures are inadequate, time-consuming, difficult, expensive, or hazardous to laboratory staff. Inherent technical limitations of PCR are present, but they are reduced in laboratories that use standardized protocols, conduct rigid validation protocols, and adhere to appropriate quality-control procedures. Improvements in PCR, especially probe-based real-time PCR, have broadened its diagnostic capabilities in clinical infectious diseases to complement and even surpass traditional methods in some situations. Furthermore, real-time PCR is capable of quantitation, allowing discrimination of clinically relevant infections characterized by pathogen replication and high pathogen loads from chronic latent infections. Automation of all components of PCR is now possible, which will decrease the risk of generating false-positive results due to contamination. The novel real-time PCR strategy and clinical applications in equine infectious diseases will be the subject of this review.