Studies On Detection Of Microsporidia (Nosema Bombycis) By Hybridization In Situ

Microsporidia ,which are eukaryotic obligate intracellular protozoon parasites, were first recognized in the last century when Nosema bombycis was found to cause the destructive pebrine disease in the commerically important, Bombyx mori. They cause severe losses to silk farms. Up to now, the detection of Microsporidia relied mainly on microscopy in sericulture. This report describes an Hybridization in situ technique for the detection of Nosema bombycis.1. Isolation of genomic DNA of Bombyx mori and Nosema bombycisThe genomic DNA of Bombyx mori can be obtained from silkworm eggs, the posterior silk glands of the 5-th instar larvae and pupae respectively. The quality and amount of DNA are eligible and sufficient for use in normal molecular biology research. Of the three materials, the posterior silk glands provide DNA of higher yield and quality than eggs and pupae.The genomic DNA of Nosema bombycis was isolated from it's spores, using two methods, ie, Alkaline-Germination and Liquid-nitrogen.2. Studies on the DNA release in situ of silkworm eggs and N.b. sporesAfter the silkworm eggs and microsporidian spores were blotted onto the hybridization membrane ,then processed by the following procedure: 1 XPBS (pH 7.8), 0.2 mol/1 NaiCOa, DNA-isolation solution, RNase A and proteinase K, their genomic DNA can release in situ while the amount of DNA was sufficient for the following hybridization in situ.3. Studies on an hybridization in situ technique of a single silkworm egg and newly-hatched silkwormA single silkworm egg, which has been inclubated for one day, three days, five days and seven days, and newly-hatched silkworm were processed by the following protocol: membrane blotting, proteinase K, NaOH, buffer I, buffer II, chloroform:isoamyl, alcohol and NaCl. Then they were hybridized in situ with a 34-base specific synthetic DNA probe. This procedure is a molecular approach for the hybridization in situ for the diagnosis of microsporidia in silkworm.4. Specific synthetic DNA probes for the detection of Nosema bombycisProbe I is a 26 -base specific synthetic DNA labeled with 32P-ATP. probe II is labeled with 32P-dATP by random primers synthesis technique with 1.2kb PCR product as template. Probe I and probe II both have good specificity when 11.5ng N.b. genomic DNA can be detected with probe I and 1.4ng with probe II. As to the optimum application amount, 4 pmol/ml is suitable for probe I while 50 ng/ml for probe II.5. Detection of microsporidia by hybridization in situ from silkworm egg and lavae infected N.b.Probe II can at least detect 296 N.b. spores when it hybridized in situ with a single egg mixed with spores. At the same time, when probe II hybridized in situ with silkworm lavae 4d, 6d and lOd after infected by N.b., they were all positive. In addtion, PCR and dotting hybridization were also used to confirm it.The result of hybridization in situ may be useful for the rapid detection of silkworm pebrine in sericulture at a single silkworm level and many samples at one time.