Rapid detection of the Marek's disease viral genome in chicken feathers by loop-mediated isothermal amplification

Abstract

A loop-mediated isothermal amplification (LAMP) method for the rapid detection of serotype 1 Marek's disease virus (MDV) was developed. The method used a set of three pairs of primers to amplify the MEQ gene for detecting serotype 1 MDV. The MDV LAMP method did not cross-react with serotype 2 and serotype 3, nor did the LAMP primers have binding sites for the common avian DNA viruses (reticuloendotheliosis virus, chicken anemia virus, subgroup J of the avian leukosis virus). Additionally, the assay could detect up to 10 copies of the MEQ gene in the MD viral genome, and it had 10 times higher sensitivity than the traditional PCR methods. The LAMP master mix was stable for 90 days at -20°C. Furthermore, the efficiency of LAMP for detection of serotype 1 MDV in clinical samples was comparable to those of PCR and viral isolation. The LAMP procedure is simple and does not rely on any special equipment. The detection of serotype 1 MDV by LAMP will be useful for detecting and controlling oncogenic Marek's disease.

Fig 1

LAMP primer designs for the detection of serotype 1 MDV. (A) Locations of LAMP primers in relation to the defined regions of the target sequence. The outer primers (F3 and B3), inner primers (FIP and BIP), and loop primers (LF and LR) are complementary to the indicated regions of the MEQ gene. FIP and BIP contain two distinct sequences (F1c + F2 and B1c + B2, respectively). (B) The positions of the LAMP primers are shown relative to the serotype 1 MDV isolate (accession no. FJ620900).

Fig 2

Comparison of the sensitivities of LAMP and PCR methods for the detection of the serotype 1 MD viral genome by 2% agarose gel electrophoresis. The plasmid containing the 180-bp MEQ gene fragment of the Ind/KA01/06 isolate was serially diluted. Each plasmid concentration (copies/tube) was subjected to both the LAMP and PCR assays. (A) LAMP assay specific to serotype 1 MDV. (B) PCR amplification of the 180-bp fragment of the MEQ gene from serotype 1 MDV. Lanes: M, 100-bp DNA marker (Genei, Bangalore, India); PC, positive control; NC, negative control.

Fig 3

Specificity of the LAMP assay in the detection of serotype 1 MDV. (A) Agarose gel electrophoresis of different serotypes of MDV. Lanes: M, 100-bp DNA marker; Serotype 3 (HVT), DNA from HVT vaccine; Serotype 2 (SB1), DNA from SB1 vaccine; Serotype 1, DNA from Ind/KA01/06 isolate. (B) Restriction enzyme analysis of the LAMP products on 3% agarose gel electrophoresis. Lanes: M, 100-bp DNA marker; 1, undigested LAMP product; 2, MboI-digested LAMP product from the Ind/KA01/06 isolate.

Fig 4

Visual detection of LAMP products for the detection of serotype 1 MDV. SYBR green I dye (1:1,000) (2 μl) was added to all the tubes after termination of the LAMP reaction. LAMP-positive reactions turned to apple green, while negative reactions remained brown. Tube 1, DNA from Ind/KA01/06 serotype 1 MDV isolate; tube 2, DNA from serotype 2 SB-1 vaccine; tube 3, DNA from serotype 3 HVT vaccine; tube 4, DNA from healthy chicken feather.