Diagnosis of Ostreid herpesvirus 1 in fixed paraffin-embedded archival samples using PCR and in situ hybridisation

In 1994, some of the high mortality episodes that affected oysters cultured in France were associated with herpesviral infections. Through histology analysis, however, viral presence could only be suspected and confirmation of histological diagnosis by transmission electron microscopy was performed in only a few cases. Subsequently, the characterisation and genome sequencing of Ostreid herpesvirus 1 (OsHV-1) made possible the development of specific molecular detection (PCR and in situ hybridisation (ISH)). Using both molecular tools, attempts were made to screen for OsHV-1 a number of fixed, paraffin-embedded oyster samples collected and processed in 1994. The aim was to compare these techniques and to estimate the accuracy of histology-based indication of viral infection. Existing DNA extraction protocols were adapted for oyster samples and two pairs of specific primers targeting small fragments (less than 200bp) were designed (C(9)/C(10) and B(4)/B(3)). The poor consistency observed between the results of PCR with both primer pairs was confirmed by statistical analysis. C(9)/C(10), which targets a repeated region of the OsHV-1 genome, appears to be the primer of choice for viral detection in archival samples. In situ hybridisation may furnish complementary information concerning the localisation of viral foci. Under certain conditions, retrospective examination of archival samples by molecular techniques may therefore provide valuable epidemiological data.     

Detection of ostreid herpesvirus 1 DNA by PCR in bivalve molluscs: a critical review

Herpes-like viral infections have been reported in different bivalve mollusc species throughout the world. High mortalities among hatchery-reared larvae and juveniles of different bivalve species have been associated often with such infections. The diagnosis of herpes-like viruses in bivalve molluscs has been performed traditionally by light and transmission electron microscopy. The genome sequencing of one of these viruses, oyster herpesvirus 1 (OsHV-1), allowed the development of DNA-based diagnostic techniques. The polymerase chain reaction (PCR) has been used for the detection of OsHV-1 DNA in bivalve molluscs at different development stages. In addition, the PCR used for detection of OsHV-1 has also allowed the amplification of DNA from an OsHV-1 variant. The literature on DNA extraction methods, primers, PCR strategies, and confirmatory procedures used for the detection and identification of herpesviruses that infect bivalve molluscs are reviewed.     

Ostreid herpesvirus 1 (OsHV-1) detection among three successive generations of Pacific oysters (Crassostrea gigas)

Ostreid Herpesvirus 1 (OsHV-1) was likely detected in Pacific oysters, Crassostrea gigas, at different stages of development. Viral infections were associated with high mortality rates in the spat and larvae. Furthermore, the persistance of OsHV-1 in asymptomatic adults was demonstrated by detection of viral DNA and proteins. In the present study, three successive generations of C. gigas (G0 and G1 parental oysters, G1 and G2 larvae) were screened for OsHV-1 by PCR. Viral DNA was detected in 2-day-old larvae, indicating that infection may take place at very early stages. Although results strengthen the hypothesis of a vertical transmission, it was not possible to predict the issue of a particular type of cross. Indeed, the detection of viral DNA in parental oysters did not systematically correspond to a productive infection or result in a successful transmission to the progeny. However, the infective status of the parents appeared to have an influence on both the infection and the survival rates of the progeny. Crosses involving an OsHV-1 infected male and a non-infected female resulted in hatching and larval survival rates statistically lower than those observed in the other types of cross. These results suggest that OsHV-1-infected females may transmit to their offspring some kind of protection or resistance against viral infection.     

Quantitative real-time PCR for detection of monkey B virus (Cercopithecine herpesvirus 1) in clinical samples

A TaqMan based real-time PCR assay was developed for rapid detection and quantitation of herpes B virus (Cercopithecine herpesvirus 1) in clinical samples. The assay utilizes B virus-specific primers and a probe to the non-conserved region of the gG gene to discriminate B virus from closely related alphaherpesviruses. Fifty copies of B virus DNA could be detected with 100% sensitivity with a wide range of quantitation spanning 6 logs. The assay was highly reproducible with intra- and inter-assay coefficients of variation of 0.6 and 2.4%, respectively. Clinical utility of the developed real-time PCR was evaluated by testing genomic DNA prepared from B virus clinical isolates (n=23) and human and monkey clinical specimens (n=62). This novel method was also compared with conventional cell culture with respect to sensitivity and specificity. TaqMan PCR assay was shown to be equally specific and more sensitive than culture method (culture vs. PCR sensitivity 50%) and was able to identify all B virus clinical isolates tested. Fast, reliable assessment of B virus DNA in infected cells and tissues makes real-time PCR assay a valuable tool for diagnosis and management of B virus infections.     

Detection of H-1 parvovirus and Kilham rat virus by PCR.

H-1 virus and Kilham rat virus (KRV) are autonomous parvoviruses which generally cause subclinical infections in rats and can cause persistent infections in cell cultures. In this study, primer sets specific for either H-1 or KRV were designed on the basis of DNA sequence comparisons of the rodent parvoviruses. The specificities of the H-1 and KRV-specific primer sets were determined by testing viral preparations of seven different parvoviruses and nine other viruses known to infect rodents. The H-1-specific PCR assay amplified the expected 254-bp product only in the presence of H-1 viral DNA and was able to detect as little as 100 fg of H-1 viral DNA. The KRV-specific PCR assay generated the expected 281-bp product only when KRV viral DNA was used as the template and was able to detect as little as 10 pg of KRV viral DNA. Each assay was able to detect its respective virus in tissues from rats experimentally infected with H-1 or KRV. In contrast, no product was amplified by either assay with tissues from mock-infected rats. Our findings indicate that these PCR assays provide rapid, specific, and sensitive methods for the detection of H-1 or KRV infection in rats and cell culture systems.     

肝螺杆菌TaqMan MGB探针实时荧光定量PCR快速检测方法的建立及应用研究

目的 建立特异、敏感、快速检测肝螺杆菌的TaqMan MGB探针实时荧光定量PCR方法.方法 针对肝螺杆菌flaB 基因的保守区设计特异性引物和探针,建立肝螺杆菌TaqMan MGB探针实时荧光定最PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008-2011年期间采集的1081份临床样本中的肝螺杆菌进行检测,同时进行分离培养和常规PCR检测.结果 建立的TaqMan MGB探针实时荧光定量PCR方法对肝螺杆菌的检测具有高度的特异性,对幽门螺杆菌、空肠弯曲菌、泰泽氏菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌均无交叉反应,检测的灵敏度达8.3拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.227,TaqManMGB探针实时荧光定量PCR效率为100％.对1081份临床样本进行检测,TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出86份肝螺杆菌阳性样本,而细菌分离培养则仅检出4份阳性.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从临床样本中检出肝螺杆菌DNA,检测时间仅为2h.结论 研究建立的TaqMan MGB探针实时荧光定量PCR方法具有可靠、特异、敏感的特点,适用于肝螺杆菌的快速检测.     

A loop-mediated isothermal amplification method for rapid detection of NDM-1 gene

A loop-mediated isothermal amplification (LAMP) method was developed for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (NDM-1), with its specificity and sensitivity having been evaluated. Six primers, including a pair of outer primers, a pair of inner primers, and a pair of loop primers, were specially designed for recognizing eight distinct sequences on the target NDM-1 gene. The amplification reaction was performed within only 40?min under isothermal conditions at 65°C in a regular water bath. The LAMP assay showed good specificity and higher sensitivity than the conventional polymerase chain reaction (PCR), with a detection limit of 1?pg genomic DNA per tube of one NDM-1-positive reference strain. The detection result for the 345 clinical samples showed 100% consistence with the result by the PCR method, and three contaminated samples could be detected correctly by LAMP assays, while they could not be detected by PCR. The LAMP method reported here demonstrated a potential and valuable means for detection of the NDM-1 gene: easy, rapid, visual, specific, accurate, and sensitive, especially useful for on-the-spot investigation.     

Specific and quantitative detection of human polyomaviruses BKV, JCV, and SV40 by real time PCR.

BACKGROUND: The polyomaviruses that infect humans, BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40), typically establish subclinical persistent infections. However, reactivation of these viruses in immunocompromised hosts is associated with renal nephropathy and hemorrhagic cystitis (HC) caused by BKV and with progressive multifocal leukoencephalopathy (PML) caused by JCV. Additionally, SV40 is associated with several types of human cancers including primary brain and bone cancers, mesotheliomas, and non-Hodgkin's lymphoma. Advancements in detection of these viruses may contribute to improved diagnosis and treatment of affected patients. OBJECTIVE: To develop sensitive and specific real time quantitative polymerase chain reaction (RQ-PCR) assays for the detection of T-antigen DNA sequences of the human polyomaviruses BKV, JCV, and SV40 using the ABI Prism 7000 Sequence Detection System. STUDY DESIGN: Assays for absolute quantification of the viral T-ag sequences were designed and the sensitivity and specificity were evaluated. A quantitative assay to measure the single copy human RNAse P gene was also developed and evaluated in order to normalize viral gene copy numbers to cell numbers. RESULTS: Quantification of the target genes is sensitive and specific over a 7 log dynamic range. Ten copies each of the viral and cellular genes are reproducibly and accurately detected. The sensitivity of detection of the RQ-PCR assays is increased 10- to 100-fold compared to conventional PCR and agarose gel protocols. The primers and probes used to detect the viral genes are specific for each virus and there is no cross reactivity within the dynamic range of the standard dilutions. The sensitivity of detection for these assays is not reduced in human cellular extracts; however, different DNA extraction protocols may affect quantification. CONCLUSION: These assays provide a technique for rapid and specific quantification of polyomavirus genomes per cell in human samples.     

Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus by loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid method for amplification of nucleic acids under isothermal conditions. In this report, a LAMP method was developed for detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV), known previously as monodon baculovirus (MBV), using a set of six primers designed to specifically recognize the PemoNPV polyhedrin gene. The optimized time and temperature conditions for the LAMP assay were 60 min at 63 °C. The sensitivity of LAMP for PemoNPV detection was approximately 50 viral copies ng⁻¹ genomic DNA (equivalent to 150 viral copies per reaction). Using a DNA template extracted from PemoNPV-infected shrimp by a viral nucleic acid kit, the detection limit of LAMP was 0.7 fg while that of nested PCR was 70 fg; therefore, the LAMP assay was 100 times more sensitive than nested PCR. The LAMP method did not amplify a product using nucleic acid extracted from shrimp infected with other viruses including yellow head virus (YHV), Taura syndrome virus (TSV), white spot syndrome virus (WSSV), Penaeus stylirostris densovirus (PstDNV) known previously as infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Penaeus monodon densovirus (PmDNV) known previously as hepatopancreatic parvovirus (HPV).     

Loop-mediated isothermal amplification assay for the diagnosis of retinitis caused by herpes simplex virus-1

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of herpes simplex virus 1 (HSV-1). The specificity of the assay was tested using DNA extracted from HSV-1-infected rabbit corneal epithelium cultures, HSV-2 grown on Vero cell line, cytomegalovirus (CMV) (AD-169), varicella zoster virus (VZV) (Oka-vaccine), adenovirus, Aspergillus flavus and Staphylococcus aureus. The specificity of LAMP was confirmed by bidirectional sequencing of the amplicons. The sensitivity of the LAMP assay was tested using different concentrations of HSV-1 DNA. To evaluate the application of the LAMP assay in clinical diagnosis, we tested vitreous samples from 20 patients with suspected viral retinitis using LAMP and real-time PCR for HSV-1. The LAMP primers amplified only HSV-1 DNA; no LAMP products were detected with the DNAs of HSV-2, CMV, VZV, adenovirus A. flavus and S. aureus. The sequences of the positive HSV-1 LAMP products perfectly (99–100%) matched the HSV-1 sequences deposited in the GenBank database. LAMP is as sensitive as real-time PCR, with the lowest detection limit being 10 copies/μL of HSV-1 DNA. Of the 20 patients with suspected viral retinitis, four tested positive for HSV-1 using real- time PCR and LAMP. A 100% concordance was observed across the two methods. The LAMP assay is a rapid, highly specific and sensitive method for the diagnosis of retinitis caused by HSV-1.     

Development of loop-mediated isothermal amplification assay for specific and rapid detection of camelpox virus in clinical samples

In this study, development of loop-mediated isothermal amplification (LAMP) assay based on ankyrin repeat protein gene (C18L) for specific and rapid detection of camelpox virus (CMLV) was carried out. The assay was optimized using viral genomic DNA (gDNA) extracted from density gradient purified CMLV and standard control recombinant DNA plasmid containing the target, which resulted in reliable amplification at 62°C for 60 min. The amplified LAMP product was identified by agarose gel electrophoresis and subsequent direct visualization under UV light or observation by naked-eye for the presence of turbidity and color change following the addition of SYBR Green I dye and hydroxy naphthol blue (HNB). The analytical specificity of LAMP and conventional PCR assays was evaluated using other related poxviruses namely buffalopox, goatpox, sheeppox, and orf viruses, which revealed only a specific amplification of CMLV. The LAMP assay was 10-fold more sensitive than the conventional PCR. Further, the assay was evaluated with DNA extracted from the cell culture isolates of CMLV (n=11) and clinical samples (n=23). These results proved that the developed LAMP is a simple, specific, sensitive, rapid and economical diagnostic tool for detection of CMLV from clinical materials.     

Rapid diagnosis of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD) in salmonid fish by a novel DNA amplification method, loop-mediated isothermal amplification (LAMP)

A new molecular diagnostic assay was developed for detection of Tetracapsuloides bryosalmonae the causative agent of proliferative kidney disease (PKD) in salmonid fish using a loop-mediated isothermal amplification method (LAMP). The PKD-LAMP assay amplifies the T. bryosalmonae DNA extracted from infected kidney, under constant temperature of 65°C within 1 h. The required equipment for DNA amplification is only a water bath. The amplification products were detected visually by using SYBR green I dye, which turns green in the presence of amplified products and remains orange in its absence, and by electrophoresis without any difference in the sensitivity of both methods. The developed PKD-LAMP assay demonstrated an exceptionally higher sensitivity than the conventional PCR. PKD-LAMP assay was found to be 100-fold more sensitive than the PCR assay. The developed assay is simple, rapid, cost-effective, specific and highly sensitive. The assay is also characterized by its field applicability, as it does not require the use of sophisticated equipment or skilled personnel.     

Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR Green chemistry

A rapid and highly sensitive real-time PCR detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single-stranded DNA virus, and white spot virus (WSV), a double-stranded DNA (dsDNA) virus infecting penaeid shrimp (Penaeus sp.), was developed using the GeneAmp 5700 sequence detection system coupled with SYBR Green chemistry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA and cycle threshold (C(T)) values over a range of 1 to 10(5) copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp beta-actin gene was amplified in parallel with the viral DNA. The C(T) values of IHHNV- and WSV-infected samples were used to determine absolute viral copy numbers from the standard C(T) curves of these viruses. For each virus and its beta-actin control, the specificity of amplification was monitored by using the dissociation curve of the amplified product. Using genomic DNA as a template, SYBR Green PCR was found to be 100- to 2000-fold more sensitive than conventional PCR, depending on the virus, for the samples tested. The results demonstrate that SYBR Green PCR can be used as a rapid and highly sensitive detection and quantification method for shrimp viruses and that it is amenable to high-throughout assay.     

Real-time PCR for detection and quantitation of leishmania in mouse tissues

Leishmania spp. are intracellular protozoan parasites that cause a wide spectrum of diseases in humans and dogs worldwide. However, monitoring of the Leishmania burden in its different hosts is still based on cumbersome and poorly sensitive methods. Here we have developed a highly accurate real-time PCR assay with which to reproducibly detect and quantify the relative Leishmania major burden in mouse tissue samples. The assay is performed with the LightCycler system using SYBR Green I and primers amplifying a ca. 120-bp fragment from minicircles of the kinetoplast DNA (kDNA). The assay was able to detect as little as 100 fg of L. major DNA per reaction, which is equivalent to 0.1 parasite. The standard curve designed for quantitation of parasites showed linearity over an at least 6-log DNA concentration range, corresponding to 0.1 to 10(4) parasites per reaction, with a correlation coefficient of 0.979. The assay also proved to have a detection range of the same magnitude as that used for detection of L. donovani and L. amazonensis, but it was 100-fold less sensitive for L. mexicana. When applied to tissues from experimentally infected mice, the real-time PCR assay is not only as sensitive as a conventional PCR assay for detection of Leishmania kDNA but also more rapid. Results indicate that this assay is compatible with the clinical diagnosis of leishmaniasis and will be a great help to scientists who use animals to monitor the efficacy of antileishmanial drugs or vaccines or decipher the unique properties of the life cycle of Leishmania spp.     

Detection of newly recognized rodent parvoviruses by PCR.

Several autonomous parvovirus isolates distinct from the prototypic rodent parvoviruses have recently been identified. These include variants of a mouse orphan parvovirus (MOPV) and a hamster isolate designated hamster orphan parvovirus (HOPV). In this study, a PCR primer set specific for these newly identified rodent parvoviruses was designed on the basis of DNA sequence comparisons of these isolates with other autonomous parvoviruses. The specificity of the primer set was determined by testing viral preparations of seven different parvoviruses and eight other viruses known to infect rodents. The PCR assay amplified the expected 260-bp product only in the presence of DNA from MOPV, HOPV, or LuIII a parvovirus of unknown species origin. The assay was able to detect as little as 10 pg of MOPV viral DNA or 1 pg of HOPV viral DNA, and it was able to detect MOPV in tissues from naturally infected mice and HOPV in tissues from experimentally infected hamsters. In contrast, the 260-bp product was not amplified from tissues of MOPV-negative mice or mock-infected hamsters. Our findings indicate that this PCR assay provides a rapid, specific, and sensitive method for the detection of MOPV in mice, HOPV in hamsters, and MOPV and HOPV in cell culture systems and that it may also be useful for the detection of LuIII contamination of cell culture systems.     

单核细胞增生李斯特菌与志贺菌双重实时PCR检测技术的建立

目的 建立一种快速、特异、灵敏、准确定量的单核细胞增生(单增)李斯特菌(Listeriamonocytogenes)与志贺菌(Shigella)同步检测方法.方法 分别根据单增李斯特菌溶血素O基因hly与志贺菌侵袭性质粒抗原H基因ipaH设计合成引物和探针.构建重组质粒pGEM-T-hly与pGEM-T-ipaH,并以EcoR I单酶切使环状重组质粒线性化作为标准品.优化反应体系,分析特异性.双重荧光定量PCR对人工污染的脱脂灭菌乳进行检测.结果 成功构建了重组质粒标准品,并运用5'、3'端分别标记FAM、TAMRA的hly基因探针和5'、3'端分别标记HEX、TAMRA的ipaH基因探针成功建立了单增李斯特菌与志贺菌同步荧光定量PCR检测方法.结论 建立的方法有较强的特异性,线性范围好(105～101copies/μl,R2≥0.998),灵敏度为10 copies/PCR,同步检测人工污染脱脂灭菌乳的灵敏度为102CFU/ml.     

Detection of Cryptococcus neoformans DNA in tissue samples by nested and real-time PCR assays

Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 x 10(1) to 2.9 x 10(4) CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections.