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Development of a new LAMP assay for the detection of CSFV strains from Cuba: a proof-of-concept study

Author:
Postel, Alexander, Pérez, Lester J., Perera, Carmen L., Schmeiser, Stefanie, Meyer, Denise, Meindl-Boehmer, Alexandra, Rios, Liliam, Austermann-Busch, Sophia, Frias-Lepoureau, Maria T., Becher, Paul
Source:
Archives of virology 2015 v.160 no.6 pp. 1435-1448
ISSN:
0304-8608
Subject:
RNA, antibodies, blood, control methods, detection limit, disease control, disease course, economic impact, enzyme-linked immunosorbent assay, genome, genotype, herds, hog cholera, loop-mediated isothermal amplification, pathogenicity, reverse transcriptase polymerase chain reaction, reverse transcription, screening, swine, vaccination, viral load, virulent strains, viruses, Cuba
Abstract:
Classical swine fever (CSF) is a devastating animal disease of great economic impact worldwide. In many countries, CSF has been endemic for decades, and vaccination of domestic pigs is one of the measures to control the disease. Consequently, differentiating infected from vaccinated animals by antibody ELISA screening is not applicable. In some countries, such as Cuba, lack of molecular techniques for sensitive, rapid and reliable detection of virus genomes is a critical point. To overcome this problem, an easy-to-use one-tube assay based on the loop-mediated isothermal amplification (LAMP) principle has been developed for detection of the genome of CSF virus (CSFV) of endemic Cuban genotype 1.4 isolates. The assay reliably detected recent isolates from three different regions of Cuba with an analytical sensitivity 10-100 times lower than that of quantitative reverse transcription RT-qPCR. Diagnostic test sensitivity was examined using reference sera from two groups of pigs experimentally infected with Cuban virulent strain CSF0705 “Margarita” and the recent field isolate CSF1058 “Pinar del Rio”. Differences in pathogenicity of the two viruses were reflected in the clinical course of disease as well as in virus loads of blood samples. Low viral RNA loads in samples from pigs infected with the field isolate caused serious detection problems in RT-LAMP as well as in RT-qPCR. Thus, it will be necessary in future research to focus on targeted sampling of diseased animals and to restrict diagnosis to the herd level in order to establish LAMP as an efficient tool for diagnosing CSF under field conditions.
Agid:
1330358