Development and Application of Quantitative Real-Time PCR for the Rapid Detection of Hemorrhagic Enteritis Virus in Tissue Samples Author(s): Jigna D. Shah , Sarah K. Scharber , and Carol J. Cardona Source: Avian Diseases, 57(2):300-302. 2013. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10384-092412-ResNote.1 URL: http://www.bioone.org/doi/full/10.1637/10384-092412-ResNote.1
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AVIAN DISEASES 57:300–302, 2013
Research Note— Development and Application of Quantitative Real-Time PCR for the Rapid Detection of Hemorrhagic Enteritis Virus in Tissue Samples Jigna D. Shah, Sarah K. Scharber, and Carol J. CardonaA Veterinary Science Building, 1971 Commonwealth Avenue, Department of Veterinary Biomedical Sciences, University of Minnesota, St. Paul, MN 55108 Received 24 September 2012; Accepted 23 January 2013; Published ahead of print 4 February 2013 SUMMARY. Hemorrhagic enteritis virus (HEV) is a type II avian adenovirus that causes intestinal hemorrhages accompanied with immunosuppression in 4-to-12-wk-old turkeys. In the present study, a hexon gene-based, quantitative real-time PCR with TaqMan probe was developed and applied to tissue samples from poultry farms to detect and quantify HEV genome copy numbers. The method was confirmed to be rapid, specific, and sensitive for the detection of HEV. This method is an excellent research and diagnostic tool that can be used to study pathogenesis and to gain insights into different phases of infection on poultry farms and for high-throughput epidemiologic investigations. RESUMEN. Nota de Investigacio´n—Desarrollo y aplicacio´n de un me´todo de PCR cuantitativo en tiempo real para la deteccio´n ra´pida del virus de la enteritis hemorra´gica en muestras de tejidos. El virus de la enteritis hemorra´gica (HEV) es un adenovirus aviar tipo II que causa hemorragias intestinales e inmunosupresio´n en pavos de cuatro a doce semanas de edad. En el presente estudio, un me´todo de PCR en tiempo real utilizando una sonda TaqMan y enfocado al gene de la proteı´na de hexo´n, se desarrollo´ y aplico´ en muestras de tejidos de granjas avı´colas, para detectar y cuantificar el nu´mero de copias del genoma del virus de la enteritis hemorra´gica. Se confirmo´ que el me´todo es ra´pido, especı´fico y sensible para la deteccio´n del virus de la enteritis hemorra´gica. Este me´todo es una excelente herramienta para la investigacio´n y diagno´stico que puede ser utilizado para estudiar la patoge´nesis y para obtener informacio´n sobre las diferentes fases de la infeccio´n en las granjas avı´colas y para realizar investigaciones epidemiolo´gicas donde se analicen nu´meros elevados de muestras. Key words: hemorrhagic enteritis virus, real-time PCR, TaqMan, spleen, turkey Abbreviations: Cq 5 threshold cycle; CV 5 coefficient of variation; HEV 5 hemorrhagic enteritis virus; LOD 5 limit of detection
Hemorrhagic enteritis is an economically important disease of 4to-12-wk-old turkeys and has occasionally occurred in 2-to-3-wk-old turkeys (3,8). It is caused by hemorrhagic enteritis virus (HEV), classified by the International Committee of Taxonomy of Viruses as Adenoviridae, Siadenovirus, Turkey adenovirus A, and Turkey adenovirus 3. Clinical signs associated with HEV infection are depression, bloody droppings resulting from intestinal hemorrhage, and sudden death 3 to 6 days postinfection. B cells are the targets of the HEV infection with clinical manifestations primarily in the intestine and spleen, consequently leading to enlargement of the spleen, i.e., splenomegaly (13). Mortality can be as high as 60%, and birds that survive the acute phase of infection have increased susceptibility to secondary bacterial infections often resulting in a secondary wave of mortality. Hemorrhagic enteritis is controlled in many countries by vaccination with an attenuated live vaccine delivered via the drinking water. The birds are generally vaccinated between 4 and 6 wk of age. The strains used for vaccination are avirulent strains of HEV or marble spleen disease virus. Diagnosis of HEV infection has been done by histopathology (6,11), virus isolation (12), serology (7), or antigen detection techniques (10). To date, only conventional, standard, or nested PCR assays for detection of HEV have been published (1,2,9). The aim of this study was to develop a real-time PCR rapid detection
Corresponding author. E-mail: [email protected]
system that is specific, sensitive, and quantitative for HEV and can be applied to tissues. MATERIALS AND METHODS Primers and probe design. The forward primer, reverse primer, and the TaqMan hydrolysis probe were designed using the National Center for Biotechnology Information Primer-BLAST program (http://www. ncbi.nlm.nih.gov/tools/primer-blast/) with ‘‘nr’’ database. The forward primer 59-GGACGGCTCCAATTGCTGG-39 and the reverse primer 59-ACAGCAAACTCTGTCCAAGGTC-39 were selected, which amplified the 95 base pair fragment of the hexon gene. A hydrolysis probe 59-CCAGCAAACTGGCCAGCGCCA-39 complementary to the internal region was selected and labeled with FAM at 59 end and BHQ1 at 39 end. The primers and probe were synthesized from Integrated DNA Technologies (Coralville, IA). Virus propagation. Turkey HEV 3 was propagated in MDTCRP19, lymphoblast cell line from ATCC (Manassas, VA) according to the method described previously (4). Cell culture supernatant was collected 3 days postinfection. Standard hexon DNA template (pUC18-B2). A plasmid pUC18 containing HEV genomic DNA, including hexon gene, was obtained (4,5). This plasmid, pUC18-B2, was used as a template for PCR optimization purposes, to generate a standard curve, and to determine the sensitivity of the assay. The conversion to copy number of the plasmid, pUC18-B2 (11,016 base pairs), in standard template plasmid DNA was done using the following equation: number of copies 5 (amount in nanograms 3 6.022 3 1023)/(length in base pairs 3 1 3 109 3 660).
Real-time PCR for hemorrhagic enteritis virus
Real-time PCR with TaqMan hydrolysis probe. Real-time PCR reactions were carried out using 7500 Real Time PCR System in the MicroAmp Optical 96 well reaction plates from Applied Biosystems (Carlsbad, CA). The amplification was performed in 25-ml reaction mixtures containing 12.5 ml of the 23 TaqMan Universal PCR Master Mix from Applied Biosystems, 100 nM of each of the primer, 100 nM of the hydrolysis probe, 0.375 ml of the ROX dye, and 10 ml of the DNA template. The PCR conditions consisted of one cycle at 95 C for 10 min and 40 three-step cycles at 95 C for 15 sec—54 C for 1 min—72 C for 35 sec. The data analysis for fractional PCR cycle for quantification or threshold cycle (Cq) values was performed using 7500 System SDS software. The parameters for the analysis were set manually to 0.00273 for the threshold, the start cycle as 3, and end cycle as 15 for baseline. These values were constant for all PCR reactions. Real-time PCR with SYBR Green. Real-time PCR reactions were carried out using the 7500 Real Time PCR System in the MicroAmp Optical 96 well reaction plates from Applied Biosystems. Amplifications were performed in 25 ml reaction mixtures containing 12.5 ml of the 23 SYBR Green II master mix, 100 nM of each of the primer, 0.375 ml of the ROX dye, and 10 ml of the DNA template. The PCR conditions consisted of one cycle at 95 C for 10 min and 40 three-step cycles at 95 C for 30 sec—54 C for 1 min—72 C for 35 sec followed by the melt curve at 60–95 C (increments of 0.2 C/sec). The melt-curve analysis was performed using 7500 system SDS software. Conventional PCR and gel analysis. Conventional PCR reactions were carried out using an MJ Research PTC-200 thermo cycler (St. Bruno, Quebec, Canada). The amplification was performed in 25-ml reaction mixtures containing 0.25 ml of the AmpliTaq Gold DNA Polymerase, 2.5 ml of 103 buffer, 2.0 ml of 25 mM MgCl2, 0.5 ml of 10 mM dNTPs, 100 nM of each of the primer, 1 ml of the 10 mg/ml bovine serum albumin (BSA) from Sigma-Aldrich (St. Louis, MO), and 10 ml of template DNA. The PCR conditions consisted of one cycle at 95 C for 10 min and 40 three-step cycles at 95 C for 30 sec—54 C for 1 min—72 C for 35 sec followed by one cycle at 72 C for 5 min. The amplified PCR products were visualized on 1.5% agarose gel and stained with ethidium bromide. HEV detection in turkey spleen samples. Samples from commercial turkeys exhibiting splenomegaly, along with splenic samples from healthy turkeys, were collected at poultry farms in different locations. The samples were stored at 280 C until processing. The processing of the samples involved aseptically mincing the splenic tissues followed by total DNA extraction using DNeasy blood and tissue kit from Qiagen (Valencia, CA), according to the manufacturer’s instructions. Each reaction involved using 10–15 mg of the spleen tissue, with modification of overnight incubation in proteinase K at 56 C. The total DNA was resuspended in 100 ml of sterile nuclease-free water, and 10 ml of a 1021 dilution was used as template in PCR reactions.
Optimization of real-time PCR conditions. Various parameters such as primer concentration (50–200 nM), probe concentration (50–200 nM), and annealing temperature (51 C–54 C) were examined over a wide range of dilutions of a standard plasmid pUC18-B2 (1024–1027) DNA template to ensure the optimal kinetics of fluorescent signal accumulation, to detect lowest Cq values, and to confirm a PCR efficiency of 95% to 105% with an R2 value $0.97. Linear regression analysis was done, and the values for R2 and PCR efficiency were calculated for each combination of parameters. The best combined R2 value and PCR efficiency were observed at both 52 and 54 C with 100 nM of each primer and 100 nM of TaqMan probe. The higher annealing temperature of 54 C, with 100 nM of each primer and 100 nM of probe, had the R2 5 0.9719, slope 5 23.256, and PCR efficiency 5 102.83% and was preferentially selected for all further PCR reactions.
Fig. 1. Amplification of the target HEV sequence and Cq values when spleen DNA, intestinal DNA, and E. coli DNA were spiked with the standard plasmid pUC18-B2 at three different concentrations of 1.375–4.375 log copies per reaction (2.38 3 101–2.38 3 104 copies per reaction).
Alternatively, to confirm the efficient use of the primers at an annealing temperature of 54 C and 100 nM of each primer, the melt-curve analysis from the real-time PCR run with SYBR Green was performed using optimized conditions. The analysis resulted in products with identical melting temperatures (78.7 C) and no primer dimers for dilutions of the plasmid template. Analytical sensitivity and specificity. To evaluate the limit of detection (LOD), a wide range of serial dilutions of plasmid pUC18-B2 (1021–10211) was used in real-time PCR reactions with the TaqMan probe. The number of plasmid copies, based on the concentration of the plasmid in each PCR reaction, was determined by using the equation described. A standard curve was generated by plotting the number of copies of plasmid vs. the Cq values, for all serial dilutions. All the reactions were run in triplicate. The LOD was found to be 1.375 log copies per reaction (2.38 3 101 copies per reaction). The linear dynamic range for the standard curve was observed to be 1.375–9.375 log copies per reaction (2.38 3 101– 2.38 3 109 copies per reaction) with PCR efficiency of 106% and an R2 value of 0.975. Both the real-time PCR and the conventional PCR detected HEV at 1.375 log copies per reaction (2.38 3 101 copies per reaction), but the LOD for the standard PCR was not determined; thus, the comparable sensitivity of tests is not known. The specificity of the primers and the TaqMan probe was confirmed with optimized real-time PCR conditions, by testing DNA from homogenized intestinal tissue of a healthy bird, splenic tissue from a healthy bird, and a culture of avian pathogenic Escherichia coli strain 01:K1:H7, which frequently coinfects birds with HEV. Amplification of the target sequences or any other PCR product was not observed in any of the above three reactions, confirming the specificity of the real-time PCR assay. However, amplification of the PCR product was observed when the DNA templates from above three sample types were spiked with the standard recombinant plasmid at 1.375 log copies per reaction (2.38 3 101 copies per reaction), which is also the LOD, confirming the same LOD in the presence of DNA from tissue samples (Fig. 1). Repeatability and reproducibility. To evaluate the repeatability and reproducibility of the assay, three sample templates (cell culture supernatant of RP19 infected with HEV, plasmid pUC18-B2, and total DNA from a HEV-infected splenic sample) and their dilutions were tested in real-time PCR reactions. Each sample was tested in triplicate in each assay to determine the intra-assay variation
J. D. Shah et al.
Table 1. Detection of HEV in spleen tissues from turkeys exhibiting clinical manifestations of infection (test farms) and turkeys with no signs of infection (control farms). Poultry farms
Log copies/10–15 mg spleen
Control farms C-A C-B C-C C-D C-E
0/4 0/14 0/7 0/1 0/40
— — — — —
Test farms T-A T-B T-C T-D T-E T-F T-G T-H
1/1 6/6 1/1 1/1 1/4 5/12 3/7 6/7
5.45 4.50–9.57 4.87 6.19 3.69 3.53–9.78 4.47–5.42 4.68–6.00
(repeatability), and the same samples were tested in three different assays to determine the inter-assay variation (reproducibility). For all the samples, the intra-assay coefficient of variation (CV) was calculated to be within the range of 3.39%–4.08%, 0.30%–0.85%, and 0.16%–1.20%, respectively. For all the samples, the interassay CV was calculated to be within the range 4.45%–6.78%, 0.20%– 2.80%, and 1.87%–3.84%, respectively. Detection of HEV in splenic samples. Splenic samples from birds (2-to-10-wk old) with clinical manifestations of HEV infection, along with birds from the same flock, were collected from several poultry farms for detection of HEV by real-time PCR. For the control group, the splenic samples of the healthy birds from poultry farms, with no signs of HEV infection, were collected. All of the samples came from birds that were vaccinated for HEV in the hatchery. The samples were processed according to the method described. All splenic samples from the control groups were negative for HEV detection (Table 1). HEV was detected in the splenic samples from the poultry farms exhibiting the clinical signs of the infection (Table 1). All the samples were also tested with conventional PCR followed by agarose gel electrophoresis. The results of from the real-time PCR data were in agreement with the conventional PCR data. DISCUSSION
The HEV infection in turkeys leads to direct and indirect economic losses because of the immunosuppressive nature of the virus. In this study, we have developed a real-time PCR rapid detection system that is rapid, specific, sensitive, and quantitative for HEV and can be applied to splenic samples from infected turkeys. Because the method is quantitative and sensitive, we were able to determine the number of viral particles in approximately 10–15 mg
of the spleen tissue sample from the birds that were heavily infected (definite clinical manifestation), as well as the birds from same flock, which had high probability of contracting the infection. The samples were also tested by conventional PCR followed by agarose gel electrophoresis. Based on the previous comparisons, the real-time the results were in agreement with the real-time PCR data. This method can be an important tool in studying the pathogenesis and epidemiology of HEV infection. REFERENCES 1. Beach, N. M., R. B. Duncan, C. T. Larsen, X. J. Meng, N. Sriranganathan, and F. W. Pierson. Comparison of 12 turkey hemorrhagic enteritis virus isolates allows prediction of genetic factors affecting virulence. J. Gen. Virol. 90:1978–1985. 2009a. 2. Beach, N. M., R. B. Duncan, C. T. Larsen, X. J. Meng, N. Sriranganathan, and F. W. Pierson. Persistent infection of turkeys with an avirulent strain of turkey hemorrhagic enteritis virus. Avian Dis. 53:370–375. 2009b. 3. Beasley, J. N., and J. Wisdom. Studies on the pathogenesis of hemorrhagic enteritis of turkeys. Avian Dis. 22:313–319. 1978. 4. Cardona, C. J., K. Nazerian, W. M. Reed, and R. F. Silva. Characterization of a recombinant fowlpox virus expressing the native hexon of hemorrhagic enteritis virus. Virus Genes 22:353–361. 2001. 5. Cardona, C. J., W. M. Reed, R. L. Witter, and R. F. Silva. Protection of turkeys from hemorrhagic enteritis with a recombinant fowl poxvirus expressing the native hexon of hemorrhagic enteritis virus. Avian Dis. 43:234–244. 1999. 6. Carlson, H. C., F. al-Sheikhly, J. R. Pettit, and G. L. Seawright. Virus particles in spleens and intestines of turkeys with hemorrhagic enteritis. Avian Dis. 18:67–73. 1974. 7. Fitzgerald, S. D., W. M. Reed, and T. Burnstein. Detection of type II avian adenoviral antigen in tissue sections using immunohistochemical staining. Avian Dis 36:341–347. 1992. 8. Harris, J. R., and C. H. Domermuth. Hemorrhagic enteritis in twoand-one-half-week-old turkey poults. Avian Dis. 21:120–122. 1977. 9. Hess, M., R. Raue, and H. M. Hafez. PCR for specific detection of haemorrhagic enteritis virus of turkeys, an avian adenovirus. J. Virol. Methods 81:199–203. 1999. 10. Ianconescu, M., E. J. Smith, A. M. Fadly, and K. Nazerian. An enzyme-linked immunosorbent assay for detection of hemorrhagic enteritis virus and associated antibodies. Avian Dis. 28:677–692. 1984. 11. Itakura, C., H. C. Carlson, and G. N. Lang. Experimental transmission of haemorrhagic enteritis of turkeys. Avian Pathol. 3:279– 292. 1974. 12. Nazerian, K., and A. M. Fadly. Propagation of virulent and avirulent turkey hemorrhagic enteritis virus in cell culture. Avian Dis. 26:816–827. 1982. 13. Suresh, M., and J. M. Sharma. Pathogenesis of type II avian adenovirus infection in turkeys: in vivo immune cell tropism and tissue distribution of the virus. J. Virol. 70:30–36. 1996.
ACKNOWLEDGMENT This work was supported by the College of Veterinary Medicine Benjamin S. Pomeroy Chair in Avian Health endowment fund at the University of Minnesota.