Detection of chronic bee paralysis virus using ultra-rapid PCR and nested ultra-rapid PCR

Abstract

The chronic bee paralysis virus (CBPV), which causes paralysis in bees, is very difficult to visually differentiate from other pathogens causing symptoms of paralysis. In addition, low numbers of viral gene molecules are difficult to detect. Therefore, this study was conducted to develop an ultra-rapid quantitative polymerase chain reaction (PCR) and nested PCR method for specifically detecting CBPV. This method is based on rapid and precise quantification of the RNA virus CBPV, using ultra-rapid reverse transcription PCR and nested PCR to overcome difficulties in detecting low numbers of viral gene molecules. Under optimal conditions, CBPV-specific ultra-rapid PCR confirmed that 1.0 × 108 CBPV-specific genes could be detected in 4 min and 17 s, and 1.0 × 102 CBPV-specific genes could be detected in 11 min and 16 s. The detection of 1.0 × 102 CBPV-specific genes was conducted by nested PCR using the inner primer set CBPV-NF/NR. High-sensitivity detection was demonstrated by confirming that the PCR product for the initial template of 1.0 × 10° CBPV-specific gene was detectable. The CBPV-specific ultra-rapid quantitative PCR and nested PCR methods are applicable for infected honey bee samples to rapidly and accurately detect low numbers of viral gene molecules. This method can be used on-site where the infected sample is collected as well as in the laboratory. This method may also be applied to detect for other honey bee pathogens with a suitable primer for each pathogen.