Loop-mediated isothermal amplification for rapid detection of Newcastle disease virus

Abstract

We have evaluated a diagnostic system based on the loop-mediated isothermal amplification (LAMP) assay for the rapid, simple, and sensitive detection of Newcastle disease virus (NDV) directly from culture isolates as well as clinical samples. By using one set of specific primers targeting the fusion protein gene, the LAMP assay rapidly amplified the target gene within 2 h, requiring only a regular laboratory water bath or heat block for reaction. The results obtained from testing the genomes of 38 NDV strains, other different viruses, and clinical samples of experimentally infected chickens showed that LAMP was as sensitive and specific as nested PCR. All LAMP-positive samples were positive by nested PCR. The LAMP assay is faster than nested PCR, cost-effective, and easy to perform. Our results clearly demonstrate that the LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of NDV infection.

FIG. 1.

(A) Schematic representation of the relationship between LAMP and nested PCR primers. The outer primers for LAMP were the same as the inner primers for nested PCR. (B) Name and sequence of each primer used in this study. The restriction site for PvuII is indicated in bold type, and the two TTTT spacers are underlined.

FIG. 2.

Sensitivities of LAMP and nested PCR methods. The LAMP and nested PCR methods were carried out using different concentrations of plasmid at picograms of plasmid per reaction. Each concentration of plasmid was tested five times. (A) LAMP reaction; (B) nested PCR reaction. Marker, 100-bp ladder size markers (Promega).

FIG. 3.

Restriction enzyme analysis of the LAMP products. Electrophoretic analysis of LAMP-amplified cDNA NDV strains and digestion with PvuII. Lane 1, undigested LAMP product; lanes 2 through 12 and 14 through 16, LAMP products after digestion with PvuII; lane 13, negative control; lane M, 100-bp ladder size markers (Promega).

FIG. 4.

Sensitivities of visual inspection and electrophoresis detection of LAMP-amplified products. Lanes N, no template; lane M, 100-bp ladder size. The number above each tube and each lane represents the dilution of LAMP product: 5×, 5-fold dilution; 10×, 10-fold dilution; 15×, 15-fold dilution; 20×, 20-fold dilution.