| The study focused on sequence feature, molecular evolution of 16SrRNA gene of Nosema bombycis, and therefore the PCR method to detect pebrine of Bombyx mori eggs was created.The 16SrRNA gene was cloned through PCR with Nosema bombycis DNA template and a special pair of primers for 16SrRNA gene of Nosema bombycis. Sequence analysis shows that the nucleotide consists of 1235bp lacking of several regions which are considered as eukaryotic sequences, and its sequence structure feature is more similar to that of prokaryotic. It was presumed that in very early stage Nosema bombycis diverged from prokaryotes. Molecular evolution analysis shows that microspores belongs to the same genus may locate different branches on phylogentic tree.The results of DNA extractions from pure microspores of Nosema bombycis demonstrate that 1.3×10-7μg DNA can be extracted from one microspore, and 3.25×10-2pg DNA, scilicet 4 microspores, in 25μl PCR system is the threshold for successful detection of PCR.The results of DNA extraction from silkworm eggs with pebrine show that total DNA normally extracted from silkworm eggs with pebrine can be used to detect pebrine among silkworm eggs when incubation eggs were prepared with 30% KOH. Practical examination research illustrates that PCR can sensitively detect pebrine from one silkworm egg, and the most sensitive for pebrine detection is one pebrine egg mixed with 300 normal eggs. The probability of identifying one pebrine egg among 100 normal eggs is about 80%. The manipulate regulation and evaluation standard of PCR examination for the pebrine ratio among silkworm eggs were created based on all of the experimental data.
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