Comparison of Loop-Mediated Isothermal Amplification and PCR for the Detection and Differentiation of Marek’s Disease Virus Serotypes 1, 2, and 3
Published: November 18, 2014
By: Grzegorz Wozniakowski,A Elzbieta Samorek-Salamonowicz, and Wojciech Kozdrun (Department of Poultry Viral Diseases, National Veterinary Research Institute, Pulawy, Poland)
Summary
The previously conducted study on loop-mediated isothermal amplification (LAMP) has shown its usefulness for the detection of Marek’s disease virus (MDV) virulent field strains. The current study improves the previously designed LAMP method with an additional pair of loop primers, which accelerates the reaction, and describes two other LAMP procedures for the specific detection of FC126 strain of turkey herpesvirus and nonpathogenic SB-1 strain. The developed LAMP procedures were also confirmed and compared with PCR. Each LAMP reaction used three pairs of specific primers designed to target the nucleotide sequence of the very virulent MDV strain, the SB-1 strain of MDV-2, and turkey herpesvirus, respectively. All LAMP reactions were flexible and provided reliable results at a wide range of incubation temperatures from 54.0 to 62.3 C in 15 to 90 min. LAMP does not need any thermocyclers, because all assays were conducted in a water bath. The green fluorescence signal was recorded under ultraviolet illumination in LAMP samples containing virulentMDV and turkey herpesvirus where SYBR Green was added to the reactionmixture, whereas the SB-1–positive samples presented orange illumination after GelRedTM staining solution. The sensitivity of the three LAMP reactions ranged from 2 log10 plaque-forming units (PFU)/ml of the virulent MDV HPRS-16 strain and turkey herpesvirus (HVT) to 3 log10 PFU/ml of the SB-1 nonpathogenic strain. The sensitivity of the compared PCR was lower by 1–2 log10 PFU/ml. The conducted studies have shown that developed LAMP methods may be used instead of PCR for the detection and differentiation of virulent and nonpathogenic MDV strains used in prophylaxis against MD. LAMP may be conducted without access to thermocyclers.
Key words: Marek’s disease, turkey herpesvirus, SB-1 strain, loop-mediated isothermal amplification
Abbreviations: CEK = chicken embryo kidney cells; dUTPase = enzyme metabolizing dUTP nucleotide to its dephosphorylated dUMP form; FC126 = nonpathogenic strain of herpesvirus of turkey used in vaccines against Marek’s disease; HPRS16 = reference, virulent MDV-1 strain; HVT = herpesvirus of turkeys; IRL = internal repeated sequence flanking unique long region; IRS = internal repeated sequence flanking unique short region; LAMP = loop-mediated isothermal amplification; MD5Marek’s disease; MDV = Marek’s disease virus; meq = main oncogene of MDV encoded by LORF7 gene; PFU = plaque-forming unit; pp24 = 24 kDa phosphoprotein encoded by MDV; TRL = terminal repeated sequences flanking unique long region; TRS = terminal repeated sequences flanking unique short region; UL = unique long region of MDV genome; US = unique short region of MDV genome; UV = ultraviolet illumination
Introduction
Marek’s disease (MD) still represents a serious threat for the commercial production of chickens, turkeys, and Japanese quail. In spite of worldwide vaccination MD presents one of the most economically important threat for poultry producers. The annual loss caused by the disease incidence reaches US$1 billion (12). MD is manifested by T-cell lymphomas causing immunosuppression, paralysis, and neurological disorders.
MD virus (MDV), belonging to the Mardivirus genus of the Herpesviridae family, is represented by three serotypes (MDV-1, MDV-2, and MDV-3 or herpesvirus of turkeys [HVT]). All pathogenic strains belong to MDV-1, and nonpathogenic MDV-2 (SB-1) and MDV-3 (FC126) strains are used in mono- and bivalent vaccines. MDV-3 has been used as a live cell-associated or lyophilized cell-free suspension since the early 1970s. MDV-2 was added to MDV-3, eliciting increased protection against MD (5,20). Vaccination againstMDprotects fromthe clinical formof the disease but does not eliminate the risk of subsequent infection with virulent strains of MDV-1. Moreover, very virulent and very virulent plus MDV strains are able to override the vaccine-induced protection (19). Losses caused by the increasing virulence of field strains has been reduced after introduction of attenuated strains based on MDV-1 like CVI988/ Rispens, an attenuated strain of the first Marek’s disease virus serotype used in vaccines against Marek’s disease.
Table 1. Sequences of primers used for LAMP and PCR assays for the detection and differentiation of MDV serotypes 1, 2, and 3. The primers are specific for the serotype indicated in primer name; for example, MDV-1—serotype 1. The nucleotides in FIP and BIP primers written in italics are thymidine linkers.
Fig. 1. Optimization of LAMP temperature. Gel electrophoresis of LAMP products for MDV-1, MDV-2, and MDV-3 detection. Temperature gradient: 1–54.0 C, 2–55.2 C, 3–56.3 C, 4–62.3 C, 5– 64.3 C, 6–65.9 C, 7–67.7 C, 8–68.0 C. In case of MDV-2 the apparent lack of the ladder products is observed at 56.3 C (MDV-2, lane 3), which is probably caused by the melting temperature of used primers or unexplained problem with UL50 amplification at this point. The differences in LAMP amplification were also observed at 54.0 C (MDV- 1, MDV-2, MDV-3, lane 1), because they have different melting points.
Because all three MDV serotypes may coexist in vaccinated and infected chickens it is important for diagnostic purposes to differentiate the serotypes and obtain a more complete overview of the epidemiology MD.
The MDV-1, MDV-2, and MDV-3 genomes consist of linear double-stranded DNA from 159 (MDV-3) to165 kb (MDV-2) and 180 kb (MDV-1). All three genomes are comprised of long and short unique sequences (UL and US, respectively) surrounded by terminal and internal inverted repeats (TRL and IRL, IRS and TRS). However, there are several unique genes for each MDV serotype. Meq oncogene, encoded by RLORF7, is exclusively present in MDV-1 strains (13). The MDV-3 genome sequence has been extensively studied. The unique long region (UL) region of MDV-3 genome is lacking chicken host range genes like MDV008 (pp24) or MDV009, but on the other hand encodes unique genes like HVT068 and HVT070 (11). This feature may be important for better understanding of differences between antigenically related serotypes of MDV, and also facilitates their differentiation. The whole-genome comparative analysis has shown that the SB-1 strain sequence is more similar to MDV-3 than to MDV-1. The UL50 region encoding enzyme metabolizing dUTP nucleotide to its dephosphorylated dUMP form (dUTPase) is a homotrimeric enzyme taking a part in nucleotide metabolism and hydrolysis of dUTP nucleotide into its dephosphorylated dUMP form. The identity of UL50 nucleotide sequence between the Md5 strain ofMDV-1 and the SB-1 strain (MDV-2) reaches 59.0%, which makes this region a good target for the differentiation between serotypes (17). Many examples of PCR or real-time PCR methods for the detection, differentiation, and quantification of MDV field and vaccine strains have been described in the last decade (2,3,9). We have previously shown that loop-mediated isothermal amplification (LAMP) can be used for the detection of MDV-1 in feather tips of infected chickens as a rapid, inexpensive, and simple molecular method that does not require advanced laboratory equipment, including thermocyclers (21). During our continuing study of LAMP for the detection of MDV-1, it has been also found that the use of new loop primers significantly accelerated the LAMP reaction, obtaining results in less than 20 min. In addition we describe the development of LAMP assays for the detection and differentiation between MDV-2 and MDV-3 strains and their comparison with PCR.
MATERIALS AND METHODS
Strains.
Ten field MDV-1 strains, 5 SB-1 MDV-2 isolates, and 10 MDV-3 isolates were isolated from vaccinated and/or infected chickens were propagated in chicken embryo kidney cells (CEKs) prepared from specific-pathogen-free (SPF) chicken embryos (LTZ, Cuxhaven, Germany) as previously described (21). MDV strains were incubated at 37.8 C in 5% CO2 until the cytophatic effect was observed. The third passage of each strain in CEKs was used for the extraction of total DNA. The MDV-1 HPRS-16 strain (8), FC126 HVT strain from commercial vaccine (Me´rial), and reference SB-1 strain (16) were also used.
Fig. 2. Sensitivity of LAMP (A), (B), and PCR (C). log10 PFU/ml 5 decimal logarithm of virus plaque forming unit per 1 ml. M—MassRuller low range DNA Ladder ready-to-use (80–1031 bp, Thermo Fisher Scientific, Waltham, MA). LAMP results were recorded as the presence of the fluorescence in tubes containing the successive dilutions of viral DNA (A). After gel electrophoresis of LAMP products the specific ladder-like pattern of bands has been observed in samples primarily considered by the tube fluorescence as the positives (B). The positive results given by PCR (C) were assumed as the presence of the single band in the successive dilutions of the virus DNA.
DNA extraction.
Total DNA was extracted from infected CEKs or reference virus stocks with the use of QIAamp Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s procedure. Briefly, 200 µl of cell suspensions were incubated with 20 µl of Proteinase K solution and AL buffer (Qiagen) in 56 C for 10 min. After incubation each solution was mixed with 200 µl of ethanol, then the solutions were centrifuged at 6000 3 g (Mikro 22R, Hettich Zetrifugen, Tuttlingen, Germany) in silica-gel–based microcolumns (Qiagen). The columns were washed with the use of AW1 and AW2 buffers and centrifuged according to manufacturer’s recommendations. DNA samples were suspended in 100 ml of AE buffer (10 mM Tris ? Cl; 0.5 mM disodium edetate, pH 9.0), frozen and stored in 270 C for testing by LAMP.
LAMP primers.
All LAMP primers were designed in Primer Explorer version 4 (NetLaboratory, Tokyo, Japan), as mentioned before (21). Three sets of specific LAMP primers for MDV-1, MDV-2, and MDV-3 were designed (Table 1). The sequences of MDV-1 outer primers (F3 and B3) and inner primers (FIP and BIP) were previously reported (21). Additionally MDV-1 loop primers (MDV-1 LF, MDV-1 LB) were also designed to accelerate the reaction. For the detection and differentiation of MDV-2 and MDV-3 strains two complete sets of LAMP primers were designed according to HVT070 gene sequence of FC126 HVT (accession number NC_002641.1) and SB-1 MDV-2 UL50 gene sequence (accession number NC_002577.1; Table 1).
LAMP optimization.
LAMP was carried out in 25 µl with the use of a polymerase mactivity isolated from thermophile Bacillus smithi DNA polymerase kit (Thermo Scientific-Fermentas, Vilnus, Lithuania), with previously described reagents and primer concentrations (21). The reaction mixture was supplemented with 1 ml of 1:10 dilution per reaction of 10,000 3 concentrated (stock solution) in dimethyl sulfoxide SYBR Green (Invitrogen) or GelRed staining dye (Biotum, Delhi, India). Reaction tubes were incubated at different temperatures in water baths ranging from 54.0 to 68.0 C and incubation periods from 15 to 90 min. Samples were observed under UV light illumination and photographed (HTC, Bellevue, WA).
PCR.
PCR was performed with the use of outer primers F3 and B3 designed for LAMP reactions. The PCR products were separated in 2% agarose gels stained with 13concentrated GelRed staining solution (Biotum).
LAMP vs. PCR.
The sensitivity of LAMP was compared with PCR. Briefly, five serial tenfold dilutions of DNA extracted from stock HPRS- 16, SB-1, and FC126 HVT were used. Serial dilutions of DNA extracted from these strains ranged from 5 log10 PFU/ml to 1 log10 PFU/ml. The fluorescence emitted by positive samples was compared to the PCR amplicon patterns separated in 2% GelRed-stained agarose gels.
RESULTS
LAMP conditions.
The optimal temperature condition of LAMP for MDV-1 was 62.3 C (Fig. 1) for 30 min when the reaction was stopped by incubation of reaction tubes for 2 min at 80 C to deactivate the polymerase. In case of MDV-2 the apparent lack of the ladder products was observed at 56.3 C (MDV-2, Fig. 1, lane 3). The differences in LAMP amplification were also observed at 54.0 C (MDV- 1, MDV-2, MDV-3, Fig. 1, lane 1). Application of loop primers (MDV- 1 LF and MDV-1 LB) for MDV-1 detection facilitated limitation of the reaction from 90 min up to 30min (data not shown). In order to confirm the observed green (MDV-1 and MDV-3) and orange MDV-2 fluorescence reaction mixtures were separated in 2% agarose gels.
Fig. 3. Detection of MDV-1, MDV-2, and MDV-3 strains isolated from vaccinated and infected chickens. The numbers indicate the following strains used in this study. Negative control—DNA extracted from noninfected SPF CEKs (No. 11 for MDV-1 and MDV-3 and No. 6 for MDV-2), M—MassRuller low range DNA Ladder ready-to-use (80–1031 bp, Thermo Fisher Scientific, Waltham, MA). The positive LAMP results were assumed on the basis of visible fluorescence of the tubes with reaction mixture under UV. The used fluorescent dyes were SYBR Green (MDV-1) and (MDV-3) (Invitrogen) or GelRed dye (MDV-2) (Biotum, Delhi, India). The specificity of the recorded fluorescence was confirmed after gel electrophoresis of LAMP products, which presented the ladder-like pattern (MDV-1, MDV-2, and MDV-3, part B).
Sensitivity and specificity of LAMP and PCR.
The detection limit of LAMP for MDV-1 and MDV-3 was 2 log10 PFU/ml; for MDV-2 the detection limit reached 3log10 PFU/ml. The results were recorded as the presence of fluorescence in tubes containing the successive dilutions of viral DNA (Fig. 2A). After gel electrophoresis of LAMP products the specific ladder-like pattern of bands has been observed in samples primarily considered by the tube fluorescence as the positives (Fig. 2B). The PCR was less sensitive, because the detection limit was 3 log10 PFU/ml and 4 log10 PFU/ml, respectively (Fig. 2C). The positive results given by PCR were assumed as the presence of the single band in successive dilutions of the virus DNA (Fig. 2C). There were no nonspecific cross-reactions among MDV-1, MDV-2, and MDV-3 LAMP assays with the use of DNA samples extracted from CEKs, which indicated the high specificity of the LAMP assays.
LAMP assay.
The optimized LAMP assay was applied for the detection and differentiation between MDV-1, MDV-2, and MDV-3 isolates from the field. (Fig. 3). After gel electrophoresis of LAMP samples the specific ladder-like pattern was observed. These results were consistent with the PCR assays (data not shown). The LAMP reactions took only 30 min and facilitated fast detection and differentiation of all three MDV serotypes. In comparison to the time needed for PCR assays the updated LAMP with use of loop primers was at least five times faster and did not require the use of a thermocycler and time-consuming agarose gel separation.
DISCUSSION
The presented study updates the previous report on the use of LAMP for specific and fast detection of MDV field strains. As we have previously presented LAMP with application of two sets of primers allowed for at least two-times-faster detection of MDV in comparison to widely used PCR (21). The usefulness of LAMP has also been confirmed by the study of Angamuthu et al. (1) who used this method for the detection of MDV in chicken feathers. Other PCR-based methods in spite of their usefulness cannot be as fast as the new variant of the LAMP technique (4,6,7,10,14,15,22). This is due to the need of use of thermocyclers or real-time PCR systems. Recently OptigeneH presented an opportunity to apply fast and reliable systems for LAMP for the real-time measurement of the exponential increase of LAMP products (18). However, the methods presented in our study facilitate detection and differentiation of MDV without requirement of the use of any thermocycling systems. The ease of detection and differentiation of MDV serotypes will have a great value for both laboratory scientists as well as field veterinarians who want to confirm the successful vaccination of birds against MD and the possible presence of virulent virus in field. Such option is given by the presented LAMP technique, which is simple, rapid, and reliable, providing important data on the actual MD epidemiological status of chicken flocks. In comparison to PCR and real-time PCR the technique is faster, simpler, and inexpensive, and hopefully will become the primary method used in veterinary laboratories.
REFERENCES
1. Angamuthu, R., S. Baskaran, D. R. Gopal, J. Devarajan, and K. Kathaperumal. Rapid detection of the Marek’s disease viral genome in chicken feathers by loop-mediated isothermal amplification. J. Clin. Microbiol. 50:961–965. 2012.
2. Baigent, S. J., L. P. Smith, R. J. Currie, and V. K. Nair. Replication kinetics of Marek’s disease vaccine virus in feathers and lymphoid tissues using PCR and virus isolation. J. Gen. Virol. 86:2989–2998. 2005.
3. Becker, Y., Y. Asher, E. Tabor, I. Davidson, M. Malkinson, and Y. Weisman. Polymerase chain reaction for differentiation between pathogenic and non-pathogenic serotype 1 Marek’s disease viruses (MDV) and vaccine viruses of MDV-serotypes 2 and 3. J. Virol. Methods 40:307–322. 1992.
4. Cai, T., G. Q. Lou, J. Yang, D. Xu, and Z. H. Meng. Development and evaluation of real-time loop-mediated isothermal amplification for hepatitis B virus DNA quantification: a new tool for HBV management. J. Clin. Virol. 4:27–276. 2008.
5. Calnek, B. W., K. A. Schat, M. C. Peckham, and J. Fabricant. Field trials with a bivalent vaccine (HVT and SB-1) against Marek’s disease. Avian Dis. 27:844–849. 1983.
6. Chen, H. T., J. Zhang, D. H. Sun, L. N. Ma, X. T. Liu, X. P. Cai, and Y. S. Liu. Development of reverse transcription loop-mediated isothermal amplification for rapid detection of H9 avian influenza virus. J. Virol. Methods 151:200–203. 2008.
7. Chena, C. M., and S. J. Cui. Detection of porcine parvovirus by loop-mediated isothermal amplification. J. Virol. Methods 155:122–125. 2009.
8. Churchill, A. E., and P. M. Biggs. Agent of Marek’s disease in tissue culture. Nature 215:528–530. 1967.
9. Islam, A., B. Harrison, B. F. Cheetham, T. J. Mahony, P. L. Young, and S. W. Walkden-Brown. Differential amplification and quantitation of Marek’s disease viruses using real-time polymerase chain reaction. J. Virol. Methods 119:103–113. 2004.
10. Ji, J., Q. M. Xie, C. Y. Chen, S. W. Bai, L. S. Zou, K. J. Zuo, Y. C. Cao, C. Y. Xue, J. Y. Ma, and Y. Z. Bi. Molecular detection of Muscovy duck parvovirus by loop-mediated isothermal amplification assay. Poult. Sci. 89:477–483. 2010.
11. Kingham, B. F., V. Zelnik, J. Kopacek, V. Majerciak, E. Ney, and C. J. Schmidt. The genome of herpesvirus of turkeys: comparative analysis with Marek’s disease viruses. J. Gen. Virol. 82:1123–1135. 2001.
12. Morrow, C., and F. Fehler. Marek’s disease: a worldwide problem. In: Marek’s disease: an evolving problem. F. Davison and V. Nair, eds. Elsevier Academic Press, London, England. pp. 49–61. 2004.
13. Nair, V., and H. J. Kung. Marek’s disease virus oncogenicity: molecular mechanisms. In: Marek’s disease: an evolving problem. F. Davison and V. Nair, eds. Elsevier Academic Press, London, England. pp. 32–48. 2004.
14. Notomi, T., H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino, and T. Hase. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28:E63. 2000.
15. Parida, M., G. Posadas, S. Inoue, F. Hasebe, and K. Morita. Realtime reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus. J. Clin. Microbiol. 42:257–263. 2004.
16. Schat, K. A., and B. W. Calnek. Characterization of an apparently nononcogenic Marek’s disease virus. J. Natl. Cancer Inst. 60:1075–1082. 1978.
17. Spatz, S., and K. Schat. Comparative genomic sequence analysis of the Marek’s disease vaccine strain SB-1. Virus Genes 42:331–338. 2011.
18. Tomlinson, J. A., M. J. Dickinson, and N. Boonham. Detection of Botrytis cinerea by loop-mediated isothermal amplification. Lett. Appl. Microbiol. 51:650–657. 2010.
19. Witter, R. L., J. M. Sharma, L. F. Lee, H. M. Opitz, and C. W. Henry. Field trials to test the efficacy of polyvalent Marek’s disease vaccines in broilers. Avian Dis. 28:44–60. 1984.
20. Witter, R. L. Increased virulence of Marek’s disease virus field isolates. Avian Dis. 41:149–163. 1997.
21. Woz´niakowski, G. J., E. Samorek-Salamonowicz, and W. Kozdrun´. Rapid detection of Marek’s disease virus in feather follicles by loop-mediated amplification (LAMP). Avian Dis. 55:462–467. 2011.
22. Yamada, Y., M. Itoh, and M. Yoshida. Sensitive and rapid diagnosis of human parvovirus B19 infection by loop-mediated isothermal amplification. Br. J. Dermatol. 155:50–55. 2006.
Wozniakowski G, Samorek-Salamonowicz E., Kozdrun W. Comparison of Loop-Mediated Isothermal Amplification and PCR for the Detection and Differentiation of Marek’s Disease Virus Serotypes 1, 2, and 3. Avian Diseases 57:539-543, 2013
Related topics:
Authors:
National Veterinary Research Institute (Poland)
National Veterinary Research Institute (Poland)
Recommend
Comment
Share
Would you like to discuss another topic? Create a new post to engage with experts in the community.
Featured users in Poultry Industry
Shivaram Rao
Pilgrim´s
PhD Director Principal de Nutrición y Servicios Técnicos de Pilgrim’s Pride Corporation
United States
United States
