Development of a direct blood-based PCR system to detect BLV provirus using CoCoMo primers

Abstract

Bovine leukemia virus (BLV), the etiologic agent of enzootic bovine leucosis, has caused pandemic outbreaks worldwide. Because transcription of the BLV is quickly blocked after infection, detecting integrated provirus at host genome is an important method of identifying whether an animal is infected. The aim of the present study was to develop a novel direct blood-based PCR system to detect the BLV provirus with high specificity and at low cost. The assay was based on the BLV-CoCoMo degenerate primers, which amplify all known BLV strains. Cattle blood samples (n = 182) were collected from the same BLV-positive farm and subjected to BLV-CoCoMo-direct-PCR to detect the BLV provirus. The proviral load was then estimated. This novel PCR method showed 100 % specificity. The BLV-CoCoMo-direct-PCR can be used in a variety of laboratory situations because it does not require expensive equipment/reagents, DNA purification, or a second round of PCR. Therefore, the method is extremely cost-effective and the risk of a false-positive result due to DNA contamination is very low.

Detection and molecular characterization of bovine leukemia virus in beef cattle presented for slaughter in Egypt

“…This could be due to the difference in the targeting genes. BLV-CoCoMo-qPCR-2 can detect even one copy of integrated provirus in the host genome and has been demonstrated to have 100 % specificity and low risk of false results [41]. The BLV infection rate found in the present study (28.0%) was higher than that reported in other previous studies in Egypt.…”

Epidemiology and genetic diversity of bovine leukemia virus

“…A variety of PCR methods, including standard PCR [ 49 , 50 ], nested PCR [ 33 , 52 , 64 ], real-time quantitative PCR (qPCR) [ 43 , 66 – 71 ], and direct blood-based PCR [ 72 , 73 ], have been extensively applied worldwide for BLV detection (Table 1 ). A variety of genes in the BLV genome are targeted for detection of BLV infection prevalence by direct diagnostic PCR methods, including the LTR region [ 43 , 70 , 71 , 73 – 77 ], and the gag [ 78 ], pol [ 69 , 79 , 80 ], env [ 55 , 79 ], and tax [ 68 , 79 ] genes.…”

“…However, the method requires expensive real-time PCR machines and reagents and involves difficult sample preparation protocols. Recently, a novel blood-based PCR system that amplifies target DNA regions without a requirement for DNA isolation and purification was developed [ 72 , 73 ]. The assay can detect BLV provirus with high specificity and at low cost, facilitating timely identification of BLV-infected cattle.…”

Molecular Epidemiological and Serological Studies of Bovine Leukemia Virus in Taiwan Dairy Cattle

“…As with all retroviruses, the integration of proviral DNA to the host genome is one of the essential steps in the BLV replication cycle. Therefore, numerous PCR-based methods were developed as highly sensitive molecular diagnosis platforms for BLV infection (13–15).…”