Importance of Primer Selection in the Application of PCR Technology to the Diagnosis of Bovine Leukemia Virus

Abstract

Abstract. The polymerase chain reaction (PCR) was used to detect bovine leukemia virus in bovine blood samples. When applied to leucocytes extracted from the blood samples, the standard method of DNA extraction gave good correlation with agar gel immunodiffusion, but a method in which 5 µl of blood was the starting material was unreliable. Selection of the primers was important, and differences in results were observed when the PCR method was applied to blood samples from different geographic areas. The sensitivity varied from 50% to 90%, depending on the primer set applied to the gag gene of proviral nucleic acid. This variation was based on geographic origin of the cattle, suggesting an influence of viral strain. In some areas, more than 1 primer may needed to optimize results.

Nested polymerase chain reaction (nPCR) based diagnosis of bovine leukemia virus in Panama

“…However, four PCR-negative samples were identified as positive with AGID, and seven nPCRnegative samples were identified as positive with ELISA, which could have several possible explanations. The most common reasons for these discrepancies have been discussed by Eaves et al (1994), who attributed them to the absence of lymphocytes in blood, and by Marsolais et al (1994), who attributed them to variations in the nucleotide sequences and a decrease in the sequences of the gag, env and pol genes as an effect of the evasion of the immune system, which was reported by Buehring et al (2014) and which, in turn, could prevent the recognition of primers at the time of banding in some viral strains or virus restrictions to the lymphoid organs as reported by Klintevall et al (1994). However, the amount of PBMC used from the collected blood (2 ml) and the methodology applied increased the availability of lymphocytes; at the same time, nPCR was more effective than other traditional PCR methodologies.…”

Using PCR for early diagnosis of bovine leukemia virus infection in some native cattle

“…The detection of BLV infection in cattle is carried out by virological methods-syncytial test (Ferrer et al, 1981;Otachel-Hawranek, 1993;Takahashi et al, 2004;Mingala et al, 2009), serological tests (Kozaczynska, 1999) and by molecular biology methods based on PCR (Naif et al, 1992;Klintevall et al, 1994;Marsolais et al, 1994;Czarnik et al, 2002;González et al, 2008). The implementation of a PCR method in BLV diagnosis could increase the detection number of infected animals.…”

“…In infected cells, BLV is integrated into host DNA in the form of a provirus, which can be detected by different molecular biology methods. Recently, polymerase chain reaction (PCR) for the detection of BLV proviral DNA has been described (Naif et al, 1992;Klintevall et al, 1994;Marsolais et al, 1994;Menendez-Arias, 2002;Czarnik et al, 2002;Amills et al, 2004). Due to some advantages of the PCR method over serological tests, many research groups developed this enzymatic reaction for BLV detection.…”

Simultaneous Use of Two Primer Pairs Increases the Efficiency of Polymerase Chain Reaction Assay in the Diagnosis of Bovine Leukemia Virus Infection

“…In only 1 other report, describing an investigation of DNA samples from BLV-infected cattle originating from various geographic regions, did the authors find markedly different results in relation to the primer pairs used. 9 The cows were examined 3 times using a different primer pair from the gag region of the BLV genome each time. The extent of divergent results obtained, is surprising in the context of the accepted low variability of the BLV genome.…”

“…However, the possible influence of the components of the amplification reaction itself on the final result was not considered. Proper selection of primers for use in the PCR is critical for objective diagnosis, and even if a particular gene is considered to be a highly conserved 9 the existence of small internal regions susceptible to mutations cannot be ignored. Simultaneous amplification in 1 tube with 2 primer pairs originating from different BLV genes minimizes the possible influence of mutations on the results of the PCR assay.…”