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Detection of koi herpesvirus DNA in tissues of infected fish 总被引：6，自引：0，他引：6
A newly recognized herpesvirus, koi herpesvirus or KHV, causes a lethal disease in common carp, Cyprinus carpio , and its colourful strain known as koi or fancy carp. In this study, we report new outbreaks of the disease, present initial characterization of the KHV genome, and describe assays for detection of KHV DNA in infected cells and tissues of infected fish. Restriction endonuclease (RE) profiles of viral DNA derived from two epidemiologically distinct KHV isolates were identical to each other. Cloned KHV BamHI and SphI DNA probes specifically hybridized to KHV DNA, but not to DNAs derived from a variety of other fish herpesviruses. The KHV DNA probes detected KHV DNA in tissues of experimentally infected koi fish by DNA hybridization. The KHV specific polymerase chain assays (PCR) were developed for rapid detection and confirmation of KHV DNA in tissues of infected fish. 相似文献
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The 16S-23S intergenic spacers (ITS) of ribosomal DNA from ten independent isolates of Streptococcus iniae and one reference strain ATCC29178 were sequenced, aligned and used to design a polymerase chain reaction (PCR) primer set for rapid and specific detection and identification of S. iniae. This primer set amplified a 377-bp DNA fragment specifically from S. iniae, but not from other common bacterial pathogens of fish or from non-fish pathogens. The PCR conditions were optimized to allow detection of the organism from agar, broth culture or infected fish tissue. The sensitivity of the PCR assay was established by the detection of DNA as low as 0.02 ng or as few as 10 CFU bacterial cells. The establishment of the specific PCR assay provides a useful tool for the identification and diagnosis of fish infection with S. iniae. 相似文献
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Lievens B Frans I Heusdens C Justé A Jonstrup SP Lieffrig F Willems KA 《Journal of fish diseases》2011,34(11):861-875
Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad-range PCR amplification combined with DNA array hybridization for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV-1, CyHV-2 and CyHV-3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were targeted. For bacterial identification, the ribosomal RNA gene was used. The developed methodology permitted 100% specificity for the identification of the target species. Detection sensitivity was equivalent to 10 viral genomes or less than a picogram of bacterial DNA. The utility and power of the array for sensitive pathogen detection and identification in complex samples such as infected tissue is demonstrated in this study. 相似文献
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Channel catfish virus (CCV) causes an acute haemorrhagic disease in channel catfish, Ictalurus punctatus (Rafinesque), fry and fingerlings. The present study describes a polymerase chain reaction (PCR)-based assay for detection of CCV DNA in the tissues of acutely infected juvenile catfish. The assay is rapid, sensitive and specifically detects CCV DNA derived from epidemiologically distinct viral isolates. The use of two independent PCR primers sets, each specific for particular CCV genes (open reading frames 8 and 59), provides a means to confirm the results and minimize false-positive results. The method identifies CCV DNA in several tissues of acutely infected fish, including the brain, blood, intestine, kidney and liver. The CCV PCR assay is useful for the diagnosis of acute CCV disease and for studies to investigate the molecular basis of CCV pathogenesis. 相似文献
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Loop-mediated isothermal amplification (LAMP) is a novel technique for nucleic acid amplification with high specificity, sensitivity and rapidity and does not require expensive equipment or reagents. In the present study, we developed and evaluated a LAMP method for the rapid detection of Renibacterium salmoninarum causing the bacterial kidney disease in salmonids. This method was more sensitive than quantitative real-time polymerase chain reaction (qPCR). Using DNA template extracted from cultured R. salmoninarum , the LAMP method gave an amplification signal from template diluted to 10−8 while the limit of detection of qPCR was10−7 . The LAMP method was also highly specific and did not amplify DNA purified from five other Gram-positive and -negative bacterial fish pathogens. The method also worked well using extracts of macrophages infected with R. salmoninarum and kidney material from rainbow trout, which were positive for R. salmoninarum by qPCR and crude R. salmoninarum culture. There was some evidence for inhibitors of the LAMP reaction in the kidney samples, which was overcome by diluting the sample. 相似文献
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Parvicapsula minibicornis is a myxosporean parasite that is associated with disease in Pacific salmon during their freshwater life history phase. This study reports the development of a quantitative (real-time) polymerase chain reaction (QPCR) to detect P. minibicornis DNA. The QPCR assay targets the 18S ribosomal subunit gene. A plasmid DNA control was developed to calibrate cycle threshold ( C T ) score to plasmid molecular equivalent (PME) units, a measure of gene copy number. Assay validation revealed that the QPCR was sensitive and able to detect 50 ag of plasmid DNA, which was equivalent to 12.5 PME. The QPCR assay could detect single P. minibicornis actinospores well above assay sensitivity, indicating a single spore contains at least 100 times the 18S DNA copies required for detection. The QPCR assay was repeatable and highly specific; no detectable amplification was observed using DNA from related myxozoan parasites. The method was validated using kidney tissues from 218 juvenile Chinook salmon sampled during the emigration period of March to July 2005 from the Klamath River. The QPCR assay was compared with histological examination. The QPCR assay detected P. minibicornis infection in 88.1% of the fish sampled, while histological examination detected infection in 71.1% of the fish sampled. Good concordance was found between the methods as 80% of the samples were in agreement. The majority of the disconcordant fish were positive by QPCR, with low levels of P. minibicornis DNA, but negative by histology. The majority of the fish rated histologically as having subclinical or clinical infections had high QPCR levels. The results of this study demonstrate that QPCR is a sensitive quantitative tool for evaluating P. minibicornis infection in fish health monitoring studies. 相似文献
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血卵涡鞭虫病是海水甲壳类的重要寄生虫病,其流行范围广、死亡率高、危害非常严重。本研究根据GenBank中已登录的血卵涡鞭虫(Hematodinium sp.)ITS1序列设计了1套引物,该引物可识别目标基因中6个不同区段。以此套引物建立了一种基于环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP)的血卵涡鞕虫病诊断方法。特异性试验结果表明,该套引物对血卵涡鞕虫检测具有较高的特异性,能有效检出血卵涡鞕虫。敏感度试验结果表明,该LAMP技术的灵敏度比常规PCR技术高4个数量级。分别运用LAMP和常规PCR技术对25份临床疑似病例进行检测,LAMP方法共检出感染病例25份,常规PCR检出感染病例23份。该技术能在65℃恒温条件下45～60min完成目的DNA的扩增,可直接通过肉眼观察反应产物中是否产生白色沉淀或经SYBRGreenI染色后通过颜色变化、扩增产物的琼脂糖凝胶电泳来定性判断结果,这将为血卵涡鞕虫病的临床诊断提供一种更加简便、快速、实用的方法。 相似文献
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Vibrio anguillarum , an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum . PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum . Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA , or from six colony-forming units (CFU) mL−1 of cultured V. anguillarum . However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g−1 tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations. 相似文献
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A nested polymerase chain reaction (PCR) system was developed for the detection of Flexibacter maritimus from fish tissue. The total procedure for the diagnosis of marine flexibacteriosis, from the point of DNA extraction to the electrophoretic analysis, can be performed in < 4 h. This was achieved by the combination of a short thermal cycling programme with a rapid DNA extraction procedure. The assay was extremely sensitive, capable of detecting as few as 75 cfu mg(-1) fish tissue. The accuracy of the nested PCR was confirmed under field conditions using tissue samples recovered during 1993-2002 from fish suffering marine flexibacteriosis. The nested PCR method proved to be efficient for the rapid and sensitive detection of F. maritimus from fish tissues and can be used for routine diagnosis of the disease caused by this pathogen. 相似文献
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Mycobacterium marinum is the causative agent of mycobacteriosis in wild and cultured fish and of atypical infection in humans. For the diagnosis of M. marinum , cultural and traditional polymerase chain reaction (PCR) methods are currently used. However, these protocols, although able to discriminate within Mycobacterium spp., have proved to be time-consuming or difficult to carry out. For this reason, the aim of this study was to obtain a rapid and specific diagnostic tool to quantify fish Mycobacterium spp. or to discriminate M. marinum from other mycobacteria. A primary PCR amplification with SYBR Green had a detection limit (dl) of 102 Mycobacterium DNA copies with a log-linear quantification range up to 104 ( R 2 = 0.99). The second PCR using FRET probes, flanking a region containing species specific nucleotide variations, was designed and validated with synthetic erp gene fragments corresponding to different mycobacterial species, different whole mycobacteria suspensions, experimentally infected fish tissues, tissues from experimentally infected fish, and samples of cultured fish. The results show that the FRET probes demonstrate a high specificity as the melting curve analysis allowed efficient discrimination of M. marinum from Mycobacterium chelonae , Mycobacterium fortuitum , Mycobacterium pseudoshottsii , Mycobacterium shottsii and Mycobacterium ulcerans . The kidney is the organ with the strongest detection signal and using fish tissues the method has a mean sensitivity of 50 DNA copies/PCR. 相似文献
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G Amagliani E Omiccioli F Andreoni R Boiani I Bianconi R Zaccone M Mancuso M Magnani 《Journal of fish diseases》2009,32(8):645-653
A multiplex polymerase chain reaction protocol for the detection of Photobacterium damselae and subspecies piscicida and damselae discrimination, with internal amplification control, was developed. Assay specificity was assessed by testing 19 target and 25 non-target pure cultures. The detection limit was 500 fg, corresponding to 100 genome equivalents. The optimized protocol was also prevalidated with spleen, kidney and blood samples from infected and uninfected sea bass, without any culture step, and it can be proposed as a valid alternative to culture standard methods for the rapid and specific diagnosis of photobacteriosis in fish. 相似文献
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The isolation of mycobacteria from field samples is problematic, and isolation of the bacterium is sometimes not even attempted. The detection of mycobacteria through traditional histology using formalin‐fixed, paraffin‐embedded (FFPE) tissues is neither sensitive nor specific. However, detection of mycobacterial DNA from FFPE specimens, suspected of being infected with mammalian mycobacteriosis, is a routine clinical procedure. In the present study, a polymerase chain reaction (PCR)‐based method was used to detect and identify mycobacteria in FFPE specimens sampled from fish suspected of being infected with fish mycobacteriosis. A total of 45 fish tissue samples, comprising of 12 tissue samples obtained from experimentally infected fish and the remainder from fish naturally infected with mycobacteria, were analysed using a PCR protocol which amplifies a fragment of the mycobacterial 65 kDa heat‐shock protein (hsp65) gene. PCR‐restriction enzyme analysis and/or sequencing were employed to further analyse the PCR amplicons. The PCR results were compared with those obtained by histology and culture. Mycobacterial DNA was detected in 34 of the 45 samples examined, of which 16 samples (47%) showed granulomatous reactions on histological examination. Using histology as the gold standard, no false‐negative PCR results were obtained. Also, considering the presence or absence of granulomas as a diagnostic criterion, the sensitivity and specificity of PCR in 42 of the FFPE tissues were 16/16 (100%) and 8/26 (～30.8%), respectively. Corresponding microbiological cultures were available for 15 cases, of which 13 were pure Mycobacterium cultures. Of these, 13 were PCR positive (100% sensitivity and 50% specificity). The PCR‐based methods used here proved sensitive, specific and rapid for the detection of mycobacteria in routinely processed paraffin wax‐embedded and formalin‐fixed histological samples, and the results of the study suggest that this method has potential use in retrospective epidemiological studies. 相似文献
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Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick 
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下载免费PDF全文 Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV‐2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV‐2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV‐2. The highly conserved ORF72 of CyHV‐2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA‐LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross‐reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA‐LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV‐2 when resources are limited. 相似文献
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The bacterial pathogen Flavobacterium psychrophilum was successfully identified from formalin-fixed, wax-embedded tissue blocks of infected rainbow trout heart and spleen tissues, using a polymerase chain reaction (PCR)-based assay. Filamentous bacteria were observed in haematoxylin and eosin and Giemsa-stained sections but no bacteria were recovered from the diseased fish using standard bacteriology isolation techniques. All infected fish had histopathological evidence of myocarditis or rainbow trout fry syndrome. Immunohistochemistry was attempted using three different anti-F. psychrophilum sera but the results were inconclusive, and an alternative molecular approach was therefore attempted. This paper describes the use of a PCR-based assay to help identify bacteria present in formalin-fixed, wax-embedded tissue samples. This is the first time that this technique has been used for the detection of fish bacteria from diagnostic samples. 相似文献
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Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), is a serious threat to salmon in aquaculture as well as to wild populations. We have developed a real-time polymerase chain reaction (PCR) for detection of Rs in kidney samples. The PCR is based on detection of unique parts of the 16S rRNA gene of Rs and DNA equivalent to 1-10 Rs genomes was detected per reaction. No cross-reactivity with other fish pathogenic or related bacteria could be demonstrated. Analysis of individual kidney samples collected from BKD classified populations identified 39.9% of the fish as positive by real-time PCR compared with 28.0% by polyclonal enzyme-linked immunosorbent assay (ELISA). The real-time PCR assay was found to be well suited for complementary use with ELISA for diagnosis of BKD, with the ability to detect clinical as well as covert Rs infections. The infection level determined by the polyclonal ELISA and by real-time PCR was significantly correlated. 相似文献
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Rapid detection and differentiation of carp oedema virus and cyprinid herpes virus‐3 in koi and common carp 下载免费PDF全文
下载免费PDF全文 Carp oedema virus (CEV) and koi herpes virus (KHV) are of major concern to common carp breeders and koi enthusiasts worldwide. The viruses cause diseases that exhibit similar external signs; thus, it is difficult to distinguish between them clinically. In this study, we developed and optimized rapid and accurate single‐ and multiplex isothermal diagnostic tools, based on recombinase polymerase amplification (RPA), for detection and differentiation of CEV and KHV. The assays were combined with a lateral flow dipstick to enable visual detection of amplification products and simplify post‐amplification analysis. Both CEV‐ and KHV‐RPA assays were specific for their target virus. The lower detection limits of the assays were similar to those of established diagnostic PCR tests for the viruses. A sample preparation method was optimized to eliminate the need for total DNA extraction from fish tissues. The estimated time to perform these RPA assays, from receiving the sample to having a result, is 50 min, compared to 10 and 7 hr for CEV‐ and KHV‐PCR tests, respectively. The assays can be performed in field situations to improve screening of fish and reduce spread of these viruses and thereby enhance the common carp and koi industries. 相似文献