sybr-green-i and Poultry-Diseases

Infectious bronchitis (IB) is a highly contagious viral disease of chickens caused by IB virus (IBV) that can cause substantial economic losses in the poultry industry. IBV variant infections have been continuously reported since the initial description in the 1930s. QX-like IBVs are the predominant circulating genotype globally. A homologous QX vaccine has superior protection efficacy compared with that of other available vaccines, and the combination of Massachusetts (Mass)-like and QX-like strains is being used to combat QX-like IBV infections. Inoculation of embryonated chicken eggs is the standard method for the titration of IBV, and the titer is expressed as 50% egg infectious dose (EID

Topics: Animals; Antibodies, Neutralizing; Bronchitis; Chickens; Coronavirus Infections; Infectious bronchitis virus; Poultry Diseases; Real-Time Polymerase Chain Reaction; Vaccines, Attenuated; Vaccines, Combined; Viral Vaccines

Waterfowl parvoviruses, including goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV), can cause seriously diseases in geese and ducks. Developing a fast and precise diagnosis assay for these two parvoviruses is particularly important.. A duplex SYBR Green I-based quantitative real-time PCR assay was developed for the simultaneous detection and differentiation of GPV and MDPV. The assay yielded melting curves with specific single peak (Tm = 87.3 ± 0.26 °C or Tm = 85.4 ± 0.23 °C) when GPV or MDPV was evaluated, respectively. When both parvoviruses were assessed in one reaction, melting curves with specific double peaks were yielded.. This duplex quantitative RT-PCR can be used to rapid identify of GPV and MDPV in field cases and artificial trials, which make it a powerful tool for diagnosing, preventing and controlling waterfowl parvovirus infections.

Topics: Animals; Benzothiazoles; Cloaca; Diamines; Ducks; Geese; Genome, Viral; Organic Chemicals; Oropharynx; Parvoviridae Infections; Parvovirus; Phylogeny; Poultry Diseases; Quinolines; Real-Time Polymerase Chain Reaction; Transition Temperature; Viral Load

A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates.

Topics: Animals; Benzothiazoles; Chickens; Coronavirus Infections; Diamines; Genotype; Infectious bronchitis virus; Lung; Nucleic Acid Denaturation; Organic Chemicals; Poultry Diseases; Quinolines; Real-Time Polymerase Chain Reaction; Trachea

Avian leukosis virus subgroup K (ALV-K) is an emerging ALV tumor virus of chickens. We developed a SYBR green-based real-time polymerase chain reaction (PCR) assay for the rapid and economical detection of ALV-K in chicken flocks. The assay was specific for ALV-K and did not cross-react with other ALV subgroup or avian influenza virus, Newcastle disease virus, or Marek's Disease virus. The method was 100 times more sensitive than conventional PCR and 10 times more sensitive than the enzyme-linked immunosorbent assay (ELISA) for the P27 antigen. The assay was also more sensitive than conventional PCR in tests of 86 clinical plasma samples. DF-1 tissue culture cells infected with 1 TCID50 ALV-K particle were identified as negative using ELISA but tested positive with the real-time PCR method. The viral loads in organs and tissues in infected chickens were highest in kidney, lungs, and glandular stomach, and these results matched ELISA findings.

Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Benzothiazoles; Chickens; Diamines; Enzyme-Linked Immunosorbent Assay; Organic Chemicals; Poultry Diseases; Quinolines; Real-Time Polymerase Chain Reaction; Viral Load

In this study, a pair of primers were designed and synthesized for the gB gene of infectious laryngotracheitis virus (ILTV) (GenBank accession number: EU104985). The recombinant plasmid was constructed as the positive reference material, and a real-time fluorescence-based quantitative PCR (RFQ-PCR) method was established to detect ILTV using synergy brands (SYBR) Green I. This method could detect 3.34 × 103 copies/μL viral nucleic acid in the initial template, the sensitivity of this method was higher than that of the conventional PCR, and the coefficient of variation (CV) in the repeatability test by this method was 3.35%. At the same time, the method was used to detect 14 suspected pathological samples for clinical analysis, and the results showed that 10 positive samples were detected, and the standard S-shaped curve was amplified. It was concluded that the RFQ-PCR method established in this study was highly sensitive, specific and repeatable, it was suitable for early clinical detection and epidemiological investigation of ILTV, and was significant in effectively controlling the occurrence and transmission of ILTV.

Topics: Animals; Benzothiazoles; Chickens; Diamines; Fluorescence; Herpesviridae Infections; Herpesvirus 1, Gallid; Organic Chemicals; Poultry Diseases; Quinolines; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity

Chickens infected with Marek's disease virus (MDV) carry the virus consistently for a long time, which increases the incidence and rate of virus-induced multi-organ tumors and increases its potential for horizontal transmission. There is a positive correlation between very virulent (vv) MDV quantity and the pathology. The purpose of this study was to determine the vvMDV loads dynamics in different phases, and the correlation between the viral quantity and tumor development. We used a SYBR Green duplex real-time quantitative PCR (q-PCR) assay to detect and quantify MDV loads and distributions in different tissues, targeting the Eco-Q protein gene (meq) of the virus and the house-keeping ovotransferrin (ovo) gene of chickens. The q-PCR was performed using different tissue DNA preparations derived from chickens which were infected with 1,000 pfu of the SDWJ1302 strain and tissue samples were collected from control and MDV-infected birds on 7, 10, 15, 21, 28, 40, 60, and 90 d post-infection (DPI). The data indicated that the MDV genome was almost quantifiable in immune organs of infected chickens as early as 7 DPI, and the number of MDV genome copies in the blood and different organs peaked by 28 DPI, but then gradually decreased by 40 DPI. The levels of viral quantity in the lymphocytes, liver, and spleen were all higher than those in other organs, and that in the feather follicles was the highest among different phases of MDV infection. The vvMDV could still be detected in peripheral blood and tissues by 90 DPI, and the vast existence of virus will stimulate tissue destruction. The data provided further evidence of viral infection involving multi-organ distribution and mainly involving immune organ proliferation, resulting in immunosuppression.

Topics: Animals; Avian Proteins; Benzothiazoles; Chickens; Conalbumin; Diamines; Herpesvirus 2, Gallid; Marek Disease; Oncogene Proteins, Viral; Organ Specificity; Organic Chemicals; Poultry Diseases; Quinolines; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Sensitivity and Specificity; Specific Pathogen-Free Organisms; Tissue Distribution; Virulence

Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease, as well as hepatitis-splenomegaly syndrome in chickens. To date, conventional reverse transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR methods have been used for the diagnosis of avian HEV infection in chickens. However, these assays are time consuming, inconvenient, and cannot detect the virus quantitatively. In this study, a rapid and sensitive SYBR Green real-time RT-PCR assay was developed to detect avian HEV RNA quantitatively in serum, liver, spleen, and fecal samples from chickens.. Based on the sequence of the most conserved HEV gene, ORF3, the primers for the assay were designed, and the standard plasmid was constructed. The detection limit of the assay was shown to be 10 copies/μl of standard plasmid/reaction, with a corresponding cycle-threshold value of 29.3. The standard curve exhibited a dynamic linear range across at least 7 log units of DNA copy number. The specificity and reproducibility of this assay was high, showing that the assay detected avian HEV RNA specifically and with little variability. Compared to conventional RT-PCR, the current assay is more sensitive for detecting avian HEV in serum, liver, spleen, and fecal samples from chickens.. A rapid, specific, and reproducible SYBR Green real-time RT-PCR assay was developed for the diagnosis of avian HEV infection in chickens. This assay can accurately detect avian HEV RNA in serum, liver, spleen, and fecal samples with more sensitivity than conventional RT-PCR.

Topics: Animals; Benzothiazoles; Chickens; Diamines; Gene Expression Regulation, Viral; Hepatitis, Viral, Animal; Hepevirus; Organic Chemicals; Poultry Diseases; Quinolines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA Virus Infections; RNA, Viral; Sensitivity and Specificity; Staining and Labeling; Viral Proteins

Avian encephalomyelitis virus (AEV) causes epidemic diseases in poultry worldwide. A SYBR Green real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay was developed for the rapid detection and quantitation of AEV in this study. A pair of specific primers was designed in the highly conserved VP1 gene of this virus. When comparing this assay with conventional RT-PCR, the rRT-PCR assay was 100 times more sensitive and could detect levels as low as 10 standard DNA copies of the AEV SX strain. The specificity of this technique was evaluated in five other avian pathogens. The AEV RNA was detected as early as three days post-infection in chicken embryos. All 18 clinical chicken brains collected from an AEV outbreak in Northwestern China were detected to be positive (100%) using the rRT-PCR assay. However, only 5 of the 18 samples were positive (28%) using the conventional RT-PCR. The results were confirmed by virus isolation in chicken embryos. This high sensitivity, specificity, and simplicity of the SYBR Green rRT-PCR approach can be a more effective method than the conventional one for AEV diagnosis and surveillance.

Topics: Animals; Benzothiazoles; Chickens; China; Diamines; DNA Primers; Encephalomyelitis Virus, Avian; Organic Chemicals; Picornaviridae Infections; Poultry Diseases; Quinolines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Staining and Labeling

Use of real-time PCR is increasing in the diagnosis of infectious disease due to its sensitivity, specificity, and speed of detection. These characteristics make it particularly suited for the diagnosis of viral infections, like avian metapneumovirus (AMPV), for which effective control benefits from continuously updated knowledge of the epidemiological situation. Other real-time reverse transcription (RT)-PCRs have been published based on highly specific fluorescent dye-labeled probes, but they have high initial cost, complex validation, and a marked susceptibility to the genetic variability of their target sequence. With this in mind, we developed and validated a SYBR Green I-based quantitative RT-PCR for the detection of the two most prevalent AMPV subtypes (i.e., subtypes A and B). The assay demonstrated an analytical sensitivity comparable with that of a previously published real-time RT-PCR and the ability to detect RNA equivalent to approximately 0.5 infectious doses for both A and B subtypes. The high efficiency and linearity between viral titer and crossing point displayed for both subtypes make it suited for viral quantification. Optimization of reaction conditions and the implementation of melting curve analysis guaranteed the high specificity of the assay. The stable melting temperature difference between the two subtypes indicated the possibility of subtyping through melting temperature analysis. These characteristics make our assay a sensitive, specific, and rapid tool, enabling contemporaneous detection, quantification, and discrimination of AMPV subtype A and B.

Topics: Animals; Benzothiazoles; Diamines; Fluorescent Dyes; Metapneumovirus; Organic Chemicals; Paramyxoviridae Infections; Poultry Diseases; Quinolines; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sensitivity and Specificity

Duck Tembusu virus (DTMUV) is a single-stranded positive-sense RNA virus that causes disease to emerge in duck flocks and results in huge economic losses to the duck industry. However, no vaccines and control measures are available in China to date. Development of reliable and fast detection methods is necessary to prevent and control this disease. Therefore, a one-step SYBR Green real-time reverse transcription polymerase chain reaction (RT-PCR) method is established here for DTMUV detection. The results show that the method can specifically detect DTMUV without cross-reactions with selected avian pathogens. The sensitivity of the assay was 1000 times greater than that of a conventional RT-PCR and able to test as few as 20 copies from RNA standard samples. The coefficients of variations of inter- and intra-assay values ranged from 0.09% to 0.36% and 0.1% to 0.23%, respectively. Testing 168 field samples and 96 experimentally infected samples by conventional RT-PCR and the one-step SYBR Green real-time RT-PCR, the positive rates were 35.1% and 73.8% from field samples and 30.2% and 64.6% from infected samples. The one-step SYBR Green real-time RT-PCR developed in this study was shown to be a sensitive, specific, high-throughput, cost-effective, and simple diagnostic tool for the rapid detection and epidemiological surveillance of the emerging DTMUV infection.

Topics: Animals; Benzothiazoles; China; Diamines; Ducks; Flavivirus; Flavivirus Infections; Organic Chemicals; Poultry Diseases; Quinolines; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

Goose circovirus (GCV) presents an immunosuppressive problem in production of geese. The infection's clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR) and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP) as a simple method of GCV detection.. The presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRed(TM) solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods.. The developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV.

Topics: Animals; Benzothiazoles; Circoviridae Infections; Circovirus; Diamines; DNA Primers; Geese; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Organic Chemicals; Poland; Poultry Diseases; Quinolines; Sensitivity and Specificity; Staining and Labeling; Temperature; Time Factors; Veterinary Medicine; Virology

A two-step SYBR-Green I-based real-time PCR with melting curve analysis was developed to detect and differentiate the avian reovirus (ARV) sigmaC gene in field and vaccine ARVs. Three primer sets were used to amplify the sigmaC gene from its 5', center, and 3' regions and analyze the melting point temperatures of nine ARVs. By combining the melting curves of the three ARV sigmaC gene regions, melting curve analysis could accurately distinguish the ARVs of different subtypes, and the results were consistent with phylogenetic analysis. The ARV sigmaC gene polymorphisms from different strains were also used to explain the differences in melting point temperatures. Compared with traditional subtyping methods, the current melting curve analysis provided an accurate test for separating ARVs, thereby making it a useful method for the improved selection of ARV vaccines.

Topics: Animals; Benzothiazoles; Capsid Proteins; Chickens; Diamines; DNA, Viral; Fluorescence; Fluorescent Dyes; Genotype; Organic Chemicals; Orthoreovirus, Avian; Phylogeny; Poultry Diseases; Quinolines; Reoviridae Infections; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Transition Temperature

Infectious laryngotracheitis is an acute viral respiratory disease of chickens with a worldwide distribution. Sensitive detection of the causative herpesvirus is particularly important because it can persist in the host at a very low copy number and be transmitted to other birds. Quantification of viral genome copy number is also useful for clinical investigations and experimental studies. In the study presented here, a quantitative polymerase chain reaction (qPCR) assay was developed using SYBR Green chemistry and the viral gene UL15a to detect and quantify infectious laryngotracheitis virus (ILTV) in ILTV-inoculated chicken embryos or naturally infected birds. The specificity of the assay was confirmed using a panel of viral and bacterial pathogens of poultry. The sensitivity of the assay was compared with two conventional PCR assays, virus titration and an antigen-detecting enzyme-linked immunosorbent assay. The qPCR developed in this study was highly sensitive and specific, and has potential for quantification of ILTV in tissues from naturally and experimentally infected birds and embryos.

Topics: Animals; Benzothiazoles; Chickens; Diamines; DNA, Viral; Herpesviridae Infections; Herpesvirus 1, Gallid; Organic Chemicals; Plasmids; Polymerase Chain Reaction; Poultry Diseases; Quinolines; Reproducibility of Results; Sensitivity and Specificity; Time Factors

This report describes a one-step real-time polymerase chain reaction assay based on SYBR Green I for detection of a broad range of duck circovirus (DuCV). Align with all DuCV complete genome sequences and other Genus Circovirus download from the GenBank (such as goose circovirus, pigeon circovirus), the primers targets to the replicate gene of DuCV were designed. The detection assay was linear in the range of 1.31 × 102-1.31 × 107 copies/μL. The reaction efficiency of the assay using the slope (the slope was -3.349) and the Y-intercept was 37.01 from the linear equation was estimated to be 0.99 and the correlation coefficient (R2) was 0.993. A series of experiments were carried out to assess the reproducibility, sensitivity, and specificity of the assay, following by the low intra-assay and inter-assay CVs for CT values obtained with the standard plasmids. The intra-assay CVs were equal or less than 1.89% and the inter-assay CVs were equal or less than 1.26%. There was no cross-reaction occurred with nucleic acids extracted from RA (Riemerella anatipestifer), E. coli (Escherichia coli), Duck Cholera (Pasteurella multocida), Avian influenza virus, avian paramyxovirus, Muscovy duck parvovirus, Duck reovirus, Duck hepatitis A virus as control templates. The nucleic acids extracted from samples of healthy ducks were used as negative controls. The assay was specific and reproducible. The established real time PCR was used to detect 45 DuCV-negative samples, which were tested using conventional PCR under the developed optimal conditions, each 15 for embryonated eggs, non-embryonated budgerigar eggs, newly hatched duck, the mixture of the lung, liver, spleen which were analysis for the presence of DuCV DNA, to conform that whether the DuCV can be transmitted vertically. Meanwhile, no positive result was shown by the real-time PCR method. The SYBR Green I-based quantitative PCR can therefore be practically used as an alternative diagnostic tool and a screening method for ducks infected with duck circovirus.

Topics: Animals; Base Sequence; Benzothiazoles; China; Circoviridae Infections; Circovirus; Diamines; DNA Primers; Ducks; Escherichia coli; Molecular Sequence Data; Organic Chemicals; Plasmids; Poultry Diseases; Quinolines; Real-Time Polymerase Chain Reaction; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Transformation, Bacterial

Goose parvovirus (GPV) is a Dependovirus associated with latent infection and mortality in geese. Currently, in a worldwide scale, GPV severely affects geese production. The objective of this study is to develop a loop-mediated isothermal amplification (LAMP) method for the sensitive, rapid, and inexpensive detection of GPV in the field.. A set of six specific primers was designed by targeting the GPV VP3 DNA. With Bst DNA polymerase large fragment, the target DNA could be amplified at 65 degrees C as early as 20 min of incubation in a simple water bath. A positive reaction was identified through the detection of the LAMP product by color change visible to the naked eye. The detection limit of the assay was 28 copies/microl of plasmid pVP3, and with equal sensitivity and specificity to fluorescent quantitative real-time PCR (FQ-PCR).. The high sensitivity, specificity, and simplicity, as well as the high throughput, make this method suitable for specific detection of GPV infection in both field conditions and laboratory settings. The utilization of complicated equipment and conduct of technical training on the GPV LAMP were not necessary.

Topics: Animals; Benzothiazoles; Dependovirus; Diamines; DNA Primers; Geese; Nucleic Acid Amplification Techniques; Organic Chemicals; Parvoviridae Infections; Poultry Diseases; Quinolines; Sensitivity and Specificity; Staining and Labeling; Temperature; Viral Structural Proteins

The objective of this study was to develop and evaluate a loop-mediated isothermal amplification (LAMP) method to detect infectious laryngotracheitis virus (ILTV) from commercial broiler and layer flocks in southern China. A set of six specific primers was designed to recognize six distinct genomic sequences of thymidine kinase (TK) from ILTV. The entire assay duration was recorded at 40 min under isothermal condition at 63.5 degrees C. The amplified products were analyzed by electrophoresis and visual judgment by the SYBR Green I dyeing. LAMP assay was 10-fold more sensitive than the routine PCR assay, with a detection limit of 46 copies per reaction. In detecting ILTV, the LAMP assay detected all 5 strains previously isolated, did not cross-react with other avian pathogens, and obtained a 100% sensitivity in 43 positive clinical samples with reference to virus isolation. Therefore, the LAMP assay may be a good alternative method for specific diagnosis of ILTV infection in primary care facilities, and in less well-equipped laboratories.

Topics: Animals; Benzothiazoles; Chickens; China; Clinical Laboratory Techniques; Diamines; DNA Primers; DNA, Viral; Electrophoresis, Agar Gel; Fluorescent Dyes; Herpesviridae Infections; Herpesvirus 1, Gallid; Nucleic Acid Amplification Techniques; Organic Chemicals; Poultry Diseases; Quinolines; Sensitivity and Specificity; Staining and Labeling; Time Factors

The current available molecular method to detect infectious bursal disease virus (IBDV) is by reverse transcriptase-polymerase chain reaction (RT-PCR). However, the conventional PCR is time consuming, prone to error and less sensitive. In this study, the performances of Sybr Green I real-time PCR, enzyme-linked immunosorbent assay (ELISA) and conventional agarose detection methods in detecting specific IBDV PCR products were compared. We found the real-time PCR was at least 10 times more sensitive than ELISA detection method with a detection limit of 0.25pg. The latter was also at least 10 times more sensitive than agarose gel electrophoresis detection method. The developed assay detects both very virulent and vaccine strains of IBDV but not other RNA viruses such as Newcastle disease virus and infectious bronchitis virus. Hence, Sybr Green I-based real-time PCR is a highly sensitive assay for the detection of IBDV.

Topics: Animals; Benzothiazoles; Birnaviridae Infections; Chickens; Diamines; Enzyme-Linked Immunosorbent Assay; Infectious bursal disease virus; Organic Chemicals; Polymerase Chain Reaction; Poultry Diseases; Quinolines; RNA, Viral; Sensitivity and Specificity

Studies were performed to determine the effects of Bcell suppression on the pathogenesis of Subgroup J avian leukosis virus (ALV-J) in broiler chickens. Neonatal chickens were treated with cyclophosphamide (CY) or PBS, and then infected with ALV-J (ADOL-7501) at 2 weeks of age. CY treatment induced B cell specific immunosuppression throughout the experiment confirmed by decreased bursal weight, intact lymphocyte mitogenetic activity stimulated by Con A and increased relative subpopulation of CD3-positive cells as measured by flow cytometry. Chickens in this experiment had Mareks disease virus exposure prior to three weeks of age as determined by the presence of lymphocytic infiltration and antibody. Virus neutralizing antibody against ALV-J was first observed at 6 weeks post-infection in some of the infected chickens in the PBS group. As expected, none of the chickens from the CY group and uninfected chickens developed virus-neutralizing antibody. The viremic status was measured by real time RT-PCR using SYBR green I dye. The percentage of viremic chickens was significantly higher, and more chickens had high titered viremia, in the CY treated group. No neoplastic foci consistent with ALVJ infection were observed in any of the experimental chickens. The frequency and intensity of viral antigen expression determined by immunohistochemistry was significantly higher in tissues from CY treated birds than those of PBS treated chickens at 3 weeks post-infection. This study showed that B cell specific immunosuppression with CY treatment in chickens resulted in increase in viremia and viral antigen load in tissues.

Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Benzothiazoles; Body Weight; Bursa of Fabricius; Chickens; Concanavalin A; Cyclophosphamide; Diamines; Flow Cytometry; Immunocompromised Host; Immunohistochemistry; Immunophenotyping; Immunosuppressive Agents; Lymphocyte Activation; Organic Chemicals; Poultry Diseases; Quinolines; Random Allocation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Spleen; Statistics, Nonparametric; Viremia

The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature (T(m)) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the T(m), which was consistently specific for the amplicon obtained; the mean peak T(m) obtained with curves specific for serotype Enteritidis was 82.56 +/- 0.22 degrees C. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 10(3) to 10(8) CFU/ml) showed good linearity (R(2) = 0.9767) and a sensitivity limit of less than 10(3) CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.

Topics: Animals; Benzothiazoles; Chickens; Culture Media; Diamines; DNA, Bacterial; Fluorescent Dyes; Organic Chemicals; Polymerase Chain Reaction; Poultry Diseases; Quinolines; Reproducibility of Results; Salmonella enteritidis; Salmonella Infections, Animal; Serotyping; Turkeys