The Danamic Changes Of Nosema Bombycis During The Growth Of Bombyx Mori Eggs Detected By PCR

Microsporidia are parasitical fungi which could infect any specises of insecta, amphibians and mammalian. Microsporidia are obligate intracellular parasites which are pathogenic microorganism that can do a huge harm in living nature. Nosema bombycis which can let the bombyx mori get pebrine could live and give birth to new N.bombycis in bombyx mori. Nosema bombycis which could spread from one to another through the air has a strong resistance in bad environment.The silkworm which is infected by the N.bombycis in the early time of the larve would die before cocooning.If the silkworm is infected by the N.bombycis in the later time of the larve, the N.bombycis could infect from the larve to the egg that could do a huge damage to the whole sericulture industry.The sericulture specialist, Ziran Huang and San Gu, have doubted the duty of vertical transmission of occurrence regularity in B.mori egg. The occurrence regularity in B.mori egg was proved by our study. It was discovered that before reverse period, the infection rate of the B.mori embryo is above 60%, but its hatching rate is 66% that strongly proved that Ziran Huang,s doubt is right. The infection of embryonic of B.mori is not according to the reversal period.The method of PCR has strong specific and highly sensitivity in the detection of N.bombycis. The PCR method was used to detect the N.bombycis before the N.bombycis have become spore already, which has an adventage in the time of detection. Once the DNA of N.bombycis has been duplicated, we could use PCR to detect it. To detect the infected time of the tissue of B.mori, the N.bombycis was injected into the five new star B.mori, and p50 was used as our test animal. 1×107 N.bombycis was injected into the new five star B.mori, after 6h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h,168h, the N.bombycis were dissected to get midgut, silk gland,gonad, malpighian tubule and epidermis. The dissected tissue was taken 100 mg in every sample and washed two times in PBS buffer. DNA of every sample were extracted in DNA kit. The concentration of DNA of every sample were detected and saved in-20?.Primer was detected by Shanghai Sangon Company. The picture of PCR result is clear and specific. The infection time of different tissue is that midgut at 24 h, silk gland at 48 h, gonad at 72 h, malpighian tubule at 72 h and epidermis at 72 h. The qPCR was used to detected the proliferation of N.bombycis at different at different time. The samples were DNA which contained the specific gene of N.bombycis. The qPCR primer were ?-GAPDH that is internal reference and?-tubulin that is target gene. The products of primer of PCR all have distinct band. The solubility curves of primer have single peak that is to say that the purity of sample was very high and was not contaminated. The amplification efficiency was about 100%. N.bombycis in midgut has a exponential growth after 24 h and reach the peak point at 144 h and 168 h.In other tissues also show the same rule.