• Journal of Apiculture
• /
• v.32 no.1
• /
• pp.27-39
• /
• 2017

PCR-chip-based ultra-rapid multiplex PCRs for detection of six major infectious pathogens in honeybee were developed. The 6 kinds of major infectious pathogens in honeybee included Paenibacillus larvae causing American Foulbrood, Melissococcus plutonius causing European Foulbrood as bacteria, Ascosphaera apis (Chalkbrood), Aspergillus flavus (Stonebrood), Nosema apis and Nosema ceranae (Nosemosis) as fungi. The developed PCR-chip-based ultra-rapid multiplex PCR showed successful amplification for all six major pathogens in the presence of more than $10^3$ molecules. The time for confirming amplification (Threshold cycles; Ct-time) was about 7 minutes for two species, and about 9 minutes for four species. Total 40 cycles of PCR took 11 minutes 42 seconds and time for melting point analysis was 1 minute 15 seconds. Total time for whole PCR detection was estimated 12 minutes 57 seconds (40 cycles of PCR and melting point analysis). PCR-chip based ultra-rapid multiplex PCR using standard DNA substrates showed close to 100% accuracy and no false-amplification was found with honeybee genomic DNA. Ultra-rapid multiplex PCR is expected to be a fast and efficient pathogen detection method not only in the laboratory but also in the apiary field.

• Journal of Apiculture
• /
• v.34 no.1
• /
• pp.39-46
• /
• 2019

BQCV multi-point PCR was developed as a rapid multiplex detection method for BQCV, one of the viral pathogens of honeybees. It could detect BQCV specific genes qualitative as well as quantitative detection based on ultra-rapid PCR. Three primer pairs (RNA dependent RNA polymerase, capsid protein, 3C like protease) were specifically designed for accurate the detection and were optimized for minimizing the detection time and increasing the sensitivity. Our advanced diagnostic system have the accuracy by lowering the concern about the variation in the BQCV detection site. In addition, it should be an opportunity to identify mutations that are mixed with other viruses.