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In vitro rearing of honey bee larvae is ideal for bioassay studies; no honey bee stable cell lines are available. Inconsistency of internal development staging of reared larvae and a susceptibility to contamination are common problems encountered. Standardized protocols on rearing larvae in vitro to make the larvae growth and development more similar to that of natural colonies are necessary to ensure the accuracy of experimental results and promote honey bee research as a model organism. Here, we concluded that when larval fasting weight was >160 mg, the time point of gut emptying can be defined as the critical point separating the larval and prepupal stages. In this way, we can conduct precise studies on the prepupal stage, such as organ remodeling during metamorphosis. Simultaneously, we further verified that recombinant AccApidaecin in genetic engineered bacteria added to the larval diet upregulated antibacterial peptide gene expression, and did not stimulate the stress response in larvae, nor did it affect the pupation rate or eclosion rate. This demonstrated that feeding recombinant AccApidaecin can enhance the individual antibacterial ability at the molecular level.
A real-time fluorescent polymerase chain reaction (PCR) assay was established to detect Streptococcus suis serotype 2. Primers and Taqman probe were designed according to cps2I (capsular polysaccharide 2I) gene using bio-software Primer Express2.0 and Oligo6.0. An 81 bp DNA fragment was amplified from S. suis serotype 2 genomic DNA, and the PCR product was cloned into pMD18-T vector and confirmed by DNA sequencing. The real-time fluorescent PCR amplification curve on a Lightcycler® showed that the method is accurate and specific for S. suis serotype 2 amplification, whereas reference bacteria S. suis, Escherichia coli, Salmonella sp., Staphylococcus aureus, Shigella sp., Listeria monocytogenes strains and a blank control were all negative. Tenfold serial dilutions of S. suis serotype 2 were used to measure the sensitivity of real-time fluorescent PCR: ten copies of bacteria could be detected in one PCR reaction and only 30 min were required for a single test. To examine the stability of the real-time fluorescent PCR, the positive control was detected at two different times. The threshold cycle (Ct) values showed no statistical differences (P>0.05). Thus, this method was stable and repeatable. These results indicate that this real-time fluorescent PCR technique could be applied for epidemic supervision in entry–exit inspection and quarantine.
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