Detection of acute bee paralysis virus and black queen cell virus from honeybees by reverse transcriptase pcr

Abstract

A reverse transcriptase PCR (RT-PCR) assay was developed for the detection of acute bee paralysis virus (ABPV) and black queen cell virus (BQCV), two honeybee viruses. Complete genome sequences were used to design unique PCR primers within a 1-kb region from the 3' end of both genomes to amplify a fragment of 900 bp from ABPV and 700 bp from BQCV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing ABPV and BQCV. Sensitivities were approximately 1,600 genome equivalents of purified ABPV and 130 genome equivalents of BQCV.

FIG. 1

Test of the RT-PCR amplification specificity of the ABPV primer set (lanes 1 through 5) and the BQCV primer set (lanes 6 through 10). Each set was tested against four different honeybee viruses. M, Pst lambda DNA marker; lane 1, ABPV; lane 2, BQCV; lane 3, CWV; lane 4, KBV; lane 5, water (negative control); lane 6, BQCV; lane 7, ABPV; lane 8, CWV; lane 9, KBV; lane 10, water (negative control).

FIG. 2

Detection of ABPV and BQCV in honeybees by RT-PCR. Extraction of the total RNA from healthy and laboratory-infected bee pupae and RT-PCR amplification were performed as described above. M, Pst lambda DNA marker; lane 1, ABPV-infected bee pupae; lane 2, healthy bee pupae; lane 3, ABPV virus stock (positive control); lane 4, water (negative control); lane 5, BQCV-infected bee pupae; lane 6, healthy bee pupae; lane 7, BQCV virus stock (positive control); lane 8, water (negative control).

FIG. 3

Determination of the sensitivity of amplification by RT-PCR of the ABPV. ABPV virus stock containing 1.6 × 10⁷ genome copies/μl was serially 10-fold diluted. One microliter of this stock was used in the RT-PCR mixture. M, Pst lambda DNA marker; lane 1, ABPV; lane 2, 10⁻¹ ABPV dilution; lane 3, 10⁻² ABPV dilution; lane 4, 10⁻³ ABPV dilution; lane 5, 10⁻⁴ ABPV dilution; lane 6, 10⁻⁵ ABPV dilution; lane 7, water (negative control).

FIG. 4

Determination of the sensitivity of amplification by RT-PCR of the BQCV. BQCV virus stock containing 1.3 × 10⁸ genome copies/μl was serially 10-fold diluted. One microliter of this stock was used in the RT-PCR mixture. M, Pst lambda DNA marker; lane 1, BQCV; lane 2, 10⁻¹ BQCV dilution; lane 3, 10⁻² BQCV dilution; lane 4, 10⁻³ BQCV dilution; lane 5, 10⁻⁴ BQCV, lane 6, 10⁻⁵ BQCV; lane 7, 10⁻⁶ BQCV dilution; lane 8, 10⁻⁷ BQCV dilution; lane 9, water (negative control).