Application of septin1 gene to detection of nosema bombycis
A technology of microsporidia and silkworm, applied in the application field of septin1 gene in the detection of microsporidia of silkworm, can solve the problems of interference with effective amplification of PCR, etc., and achieve the effect of strong specificity, high specificity and high sensitivity
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Embodiment 1
[0037] Example 1 septin1 Gene function research
[0038] The inventor is developing the silkworm microsporidia N. bombycis (Guangdong strain) on the basis of transcriptomics research, the results of transcriptome sequencing analysis identified for the first time that it may be related to N. bombycis related to infection and reproduction Septins genes, named septin1 (Accession: KF421133.1), septin2 (Accession: KJ451481.1), septin3 (Accession: KJ451482.1). The present embodiment is infected with the silkworm Microsporidia N. bombycis Silkworm larvae, eggs and other different developmental stages were carried out septin1 Analysis of expression differences.
[0039] The test method is: silkworms from the 4th instar (bombyx mori variety: Liangguang No. 2) are fed with No. spp. silkworm, once in the morning and once in the evening, and then normally reared. Every day after the poisoning (from the first day of the fourth instar to the fifth instar mature silkworms), t...
Embodiment 2
[0047] Example 2 Septin1 Application of the gene as a target gene to detect whether silkworm eggs are infected with Microsporidium mori
[0048] (1) Extraction of total DNA from silkworm eggs
[0049] Genomic DNA was extracted using the DNeasy Plant mini kit produced by QIAGEN, and the steps were as follows:
[0050] A. Take 20 silkworm eggs and put them into a mortar, grind them thoroughly with liquid nitrogen, and collect the ground powder into a 1.5mL centrifuge tube;
[0051] B. Add 400 μL AP1 and 4 μL RNase, and vortex to mix (400 μL APL and 4 μL RNase should not be mixed before use); after mixing, incubate at 65°C for 10 min (during this period, invert the test tube up and down for 2-3 Second-rate);
[0052] C. Add 130 μL AP2, mix and ice-bath for 5 minutes; then centrifuge at 14 000 rpm for 5 minutes;
[0053] D. Draw the supernatant into the collection tube in the QIA shredder spin column, centrifuge at 14 000rpm for 2min;
[0054] E. Transfer the supernatan...
Embodiment 3 Embodiment 2
[0066] The specific detection of the primer described in embodiment 3 embodiment 2
[0067]Take 30 "poisonous" silkworm eggs, healthy silkworm eggs and purified Nosporum silkworm respectively, extract total DNA according to the method in Example 2, then carry out PCR amplification, and use agarose gel electrophoresis to detect the results. Test results such as figure 1 As shown: DNA fragments of 699 bp were detected in "toxic" silkworm eggs and purified No. The septin1R primer amplifies the N.b specific target band of about 699 bp, and there are no other bands with sizes different from the target fragment. Therefore, the septin1 As the target gene for detection, the inhibitory substances in the silkworm egg extract will not interfere with the effective PCR amplification of pathogenic gene DNA, and the detection results are more accurate and sensitive. It can quickly detect whether it is infected with No. spp. silkworm. The above results show that the primers described in E...
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