A simple rapid colorimetric reaction based post PCR analysis of Marek’s disease virus in chicken

Abstract

Marek’s disease is an oncogenic virus that produces malignant lymphomas in chickens. Rapid easy detection of Marek’s disease is of great importance for early diagnosis and control of the disease. In this study, identification of Marek’s disease virus serotype 1 was done by using a colorimetric integrated polymerase chain reaction with Meq gene. The forward primer of the Meq gene which is specific to MDV serotype 1 was integrated with HRPzyme sequence (916 bp) and amplified during the PCR. The PCR product including HRPzyme produced the distinguished blue colour with chromogenic substrates which was visualized by the naked eye. From these observations, simple, rapid colorimetric PCR assay of Marek’s disease virus was performed without using agarose gel electrophoresis.

Introduction

Marek’s disease (MD) is an infectious re-emerging viral disease of chickens caused by Marek’s disease virus (MDV), Gallid herpes virus 2 [1, 2], inducing neoplastic infections in poultry in the form of T-cell lymphomas in the visceral organs and peripheral nerves [3]. Marek’s disease virus belongs to the Herpesviridae family. Since the horizontal route only is the way of transmission in this disease, the virus is highly stable in dry feathers and dust [4]. Serotypes of MD includes serotype 1 (Gallid herpes virus 2) which causes MD, serotype 2 (Gallid herpes virus 3) and serotype 3 (Maleagrid herpes virus) both are avirulent which are used for vaccination against MD.

MDV genome encodes more than 200 genes and several genes unique for oncogenic MDVs have been identified to date. The MDV gene encodes a major lytic and transformation-maintaining phosphoprotein antigen (pp8) together with Marek’s EcoRI-Q-encoded protein (MEQ) and virus- induced IL−8 homology (vIL−8) genes that were reported to play a role in viral oncogenicity and pathogenicity [5].

Polymerase chain reaction has been used for the rapid detection of MDV strains [6] and it can be used to identify the trace amount of target DNA in an exponential manner. Amplified PCR products are typically detected by using agarose gel electrophoresis [7] or fluorescent dyes [8] and these procedures require special appliances and they are time- consuming and costly.

In this above mentioned limitations, it is more important for alternative methods that can be used for rapid and easy identification of amplified PCR products without using complicated instruments. New DNA sensing platforms based on molecular beacons [9] and nanoparticle-tagged probes [10, 11] are developed to target this goal but these reported techniques require the use of fluorescence conjugation or probe thiolation procedures, both of which are costly and time- consuming [12]. In this research, a new rapid colorimetric PCR assay to detect MDV serotype I in chicken was developed.

Materials and methods

Primer designing

The PCR primers specific to MDV serotype−1 targetting Meq oncogene used in this study was already published [13].

Primer designing for colorimetric PCR

The integration of the anti HRPzyme sequence (60 bp) along with the conventional PCR (856 bp) forward primer of serotype 1 and reverse primer without antiHRPzyme integration were used in this reaction. The expected amplicon size of colorimetric PCR was 916 bp.

Virus source and DNA isolation

Cell culture adopted MDV serotype I available in the Department of Animal Biotechnology, Madras Veterinary College, Chennai-600 007, India was utilized in this study. The commercially available serotype 2 live vaccine (SB1 + HVT combined bivalent) and serotype 3 vaccine (HVT only, monovalent) were purchased from Namakkal district, Tamil Nadu, India. The DNA was extracted using a DNA extraction kit (QIAGEN, USA) from the Cell culture adopted MDV serotype I, cell associated vaccine (serotype 2/3), and lyophilized vaccines (serotype3) as per the protocol given in the kit.

Optimization of colorimetric PCR

The colorimetric primers were checked initially with DNA isolated from Cell culture adopted MDV serotype I. The parameters such as temperature (Tm) value, DNA concentration, and Primer concentration ranged from 62˚C to 67˚C, 0.5ng/µl to 30ng/µl, 0.1pmol/µl to 5pmol/µl, respectively, and were used for the initial optimization of colorimetric PCR for serotype 1. The optimized values were found to be 64˚C for Tm value, 5 ng/µl for DNA concentration, 3pmol/µl for primer concentration.

Specificity of colorimetric PCR primers with different serotypes of MDV

Serotype 1 specific colorimetric primers were cross checked with other serotypes of MDV.

Specificity of colorimetric PCR primers with other oncogenic viruses

MDV Serotype 1 primer was cross checked with other oncogenic viruses of Avian Leukosis Virus (ALV) and Reticuloendotheliosis Virus (REV) under the optimized PCR conditions. Avian Leukosis Virus (ALV) and Reticuloendotheliosis Virus (REV) samples were received from Department of Animal Biotechnology, Madras Veterinary College, Chennai-7, Tamil Nadu, India.

Sequencing

The positive PCR products were sequenced by bidirectional cycle sequencing (Eurofins company, Bengaluru, India). The obtained sequences were confirmed for homology by Basic Local Alignment Search Tool (BLASTn).

Colorimetric PCR reaction

The steps include 25 µL PCR products were added into the mixture including 2 µL KCl (50 mM), 4 µL HEPES buffer, 2 µL hemin (45 µM), and the solution were incubated at 37º C for 15 min. Then 30µL of freshly prepared TMB reagent (1 mg/ml) and 5 µl of 30% H₂O₂ was quickly added to the above mentioned mixture and incubated at room temperature for 10 min.

Optimization of colorimetric reaction

In the colorimetric analysis during optimization different concentrations 35, 40, 45, 50, 55, 60, 65, 70, 75 μm, KCl concentration 10, 20,30, 40, 50, 60, 70, 80, 90, 100 mM, H₂O₂ 30%, 15%, 10%, 5%, 1%, 0.1% and TMB concentration 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.1 mg/ml for the optimization of color change in the reaction.

Results

The expected amplicon sizes were observed in 856 bp for conventional primers and 916 bp for colorimetric primer (Fig. 1) of serotype 1 in 1.5% agarose gel.

Fig. 1.

Conventional and colorimetric PCR amplification of positive MDV sample

Optimization of colorimetric PCR parameters

The optimized values of colorimetric PCR were found to be 64˚C for Tm value, 5ng/µl for DNA concentration, and 3 pmol/µl for primer concentration (Fig. 2).

Fig. 2.

Optimisation of Tm value, DNA concentration and primer concentration for colorimetric PCR

Specificity of colorimetric reactions

Mareks disease virus (MDV) serotype 2 and 3 did not show amplification with the colorimetric primers used in this study (Fig. 3). MDV serotype 1 specific primers did not amplify the other oncogenic viruses i.e. ALV and REV also (Fig. 4).

Fig. 3.

Specificity of colorimetric PCR primers with serotype 2 and 3

Fig. 4.

Specificity of colorimetric PCR primers with ALV and REV

Optimization of colorimetric reaction

Colorimetric PCR products could induce the color change of the solution based on the HRPzyme-like catalytic property of the G-quadruplex / hemin complex. So it is essential to optimize the concentration of hemin in the colorimetric reaction. In the colorimetric analysis during optimization different concentrations of hemin ranges from 35, 40, 45, 50, 55, 60, 65, 70, 75 μm, and different concentrations of KCl ranges from 10, 20, 30, 40, 50, 60, 70, 80, 90, 100mM were initially used (Fig. 5). From this research, 45 μm concentration of hemin and 50 mM concentration of KCl were produce better color change (Fig. 6) in the reaction. Negative control revealed no colour.

Fig. 5.

Colorimetric reaction parameters optimization

Fig. 6.

Colorimetric PCR-Blue colour positive reaction

Cut off values

Cut off values of positive samples: >0.107 ± 0.15 at 450 nm Cut off values of negative samples: <0.071 ± 0.01 at 450 nm.

Specificity of colorimetric reactions

Serotype 1 specific PCR integrated colorimetric assays were cross checked with other serotypes of MDV and other oncogenic viruses of ALV and REV (Fig. 7) under the optimized colorimetric conditions.

Fig. 7.

Specificity of colorimetric reactions with other serotypes, ALV and REV

Sensitivity of colorimetric reactions

The sensitivity of assay has been shown the positivity up to 1ng/µl DNA template concentration, while conventional PCR detect up to 5ng/µl DNA template concentration.

Screening of field samples

Samples were collected from different poultry farms in and around Namakkal, other Districts of Tamil Nadu, Andhra Pradesh, Maharashtra and Puducherry. Out of 430 samples screened for MDV by calorimetric PCR as well as conventional PCR, 194 samples showed positivity by calorimetric PCR while 188 samples showed positivity by conventional PCR.

Discussion

Marek’s disease virus is subdivided into three serotypes viz. Serotype1 (MDV−1), serotype2 (MDV−2) and serotype 3 (MDV−3). Differentiation of these serotypes by molecular techniques is very important for the control of the disease [14]. The previous report on the differentiation of pathogenic and nonpathogenic MDVs was based on the amplification of tandem direct repeats of 132 bp sequence within the MDV gene [15].

Serotype 1 poses a serious threat to the poultry industry in the way of economic losses throughout the world [16, 17]. Rapid detection of pathogenic MDV virus by using various polymerase chain Reactions has been used [6, 18]. PCR is the method of choice for detection of Marek’s disease virus. Amplified PCR products are typically detected by using agarose gel electrophoresis [7] or fluorescent dyes [8] and these procedures are required complicated instrumentation, time consuming or costly. In the present research, amplified PCR products were detected easily and directly by adding the amplified PCR products into the catalytic system. The amplified PCR product induced the blue colour change of the solution due to HRPzyme-like catalytic property of the G-quadruplex/hemin complex compared to negative control. The optical signals has been tried by the naked eye and distinguished from the negative control. Similar optical signals by the way of blue colour production for the rapid and easy detection of Vibrio parahaemolyticus [19] and other pathogens Salmonella typimurium, Escherichia coli, Listeria monocytogenes, and Bacillus cereus [20] were reported successfully.

The methodology was developed for the detection of serotype 1 MDV by colorimetric PCR primers consisting of target template DNA of serotype 1 Meq gene, and a specific sequence (anti- HRPzyme) which is complementary to the signal reporting. Using these forward and normal reverse primers in the presence of target DNA of MDV serotype 1 the amplified double standard PCR product is having HRPzyme sequence presented in excess amount. From these peculiar PCR products will be catalyzed the complex oxidation of TMB by the formation of HRPzyme -Hemin complex in the presence of Hemin producing a blue coloured catalyzed product and it was visualized by the naked eye. Based on the blue color development indicates the positivity for serotype 1MDV and the negative sample did not show a colour reaction. The developed assay did not show any cross reactivity with other oncogenic viruses which indicates the specificity of the assay. Further, it was also confirmed by conventional PCR with serotype 1 MDV.

In general, post PCR analysis requires gel casting, electrophoresis run, gel staining and gel visualization. In this analysis, maximum 10–20 samples can be analyzed at a time in standard electrophoresis gel and 96 samples only can be amplified in a standard thermal cycler at the same time. From these points, for rapid result in a resource-poor field level diagnosis in outbreak cases or short time handling of large number of samples in well equipped laboratories [21], this assay analysis will be useful in a low cost post PCR analysis of MDV in chicken. In this regard, the development of a colorimetric assay based result reading platform and application in PCR based diagnostics was utilized in this study for the diagnosis of serotype 1 MDV to prevent further transmission of the disease as well as outbreak [22].

The colorimetric PCR products induce the color only in serotype 1 whereas other samples could not produce the color. All these results indicate that colorimetric integrated PCR could be used in the rapid and easy post PCR analysis of MDV serotype 1 with expected specificity and without any cross reactivity. The usage of colorimetric PCR for commercial purposes should be validated in the future with other techniques available.

Conclusion

Rapid easy detection of Marek’s disease is need of an hour for early diagnosis and control of the disease. The PCR product including HRPzyme produced the distinguished blue colour with chromogenic substrates which was visualized by the naked eye. In this study, a simple, rapid colorimetric PCR assay of Marek’s disease virus was performed without using agarose gel electrophoresis.