外文摘要：NOVELTY - Detecting seed carrying bacteria based on sol density gradient centrifugation comprises e.g. preparing rice seed to be detected, taking 50 seeds of rice to be detected, firstly cleaning the rice seed to be detected for 1-2 times by 200 ml of sterilized water, orderly using 70% ethanol 2.5 ml to soak for 2-3 minutes, soaking by 2.5% 2.5 ml sodium hypochlorite, soaking for 3-5 minutes, by 70% of ethanol 2.5 ml for 20-30 seconds, using sterilized water to wash the seeds for 3-4 times, after airing seeds, grinding in sterile mortar, filtering with 50 mesh sieve and grinding.
USE - The method is useful for detecting seed carrying bacteria based on sol density gradient centrifugation (claimed).
DETAILED DESCRIPTION - Detecting seed carrying bacteria based on sol density gradient centrifugation comprises (i) preparing rice seed to be detected, taking 50 seeds of rice to be detected, firstly cleaning the rice seed to be detected for 1-2 times by 200 ml of sterilized water, orderly using 70% ethanol 2.5 ml to soak for 2-3 minutes, soaking by 2.5% 2.5 ml sodium hypochlorite, soaking for 3-5 minutes, by 70% of ethanol 2.5 ml for 20-30 seconds, using sterilized water to wash the seeds for 3-4 times, after airing seeds, grinding in sterile mortar, filtering with 50 mesh sieve, grinding the rice seed for 50 meshes to sieve 100 meshes sieve for filtering, taking rice seeds under 100 meshes sieve into a sterilized culture dish whose diameter is 9 cm, (ii) preparing of Ludox HS-40 (RTM: Dioxosilane) isotonic mother liquor with 10x phosphate-buffered saline buffer solution and Ludox HS-40 (RTM: Dioxosilane) of ratio 1: 9 prepared by mixing, with liquor density is 1.308 g/ml, (iii) preparing continuous density Ludox HS-40 (RTM: Dioxosilane) separating the working solution i.e. separating the working solution, using 10x phosphate-buffered saline buffer solution Ludox HS-40 (RTM: Dioxosilane), isotonic with 9 ml mother liquor to prepare the solution, namely separating the working solution Ludox HS-40 (RTM: Dioxosilane), separating working liquid, using 10x phosphate-buffered saline buffer solution of 4 ml with 6 ml Ludox HS-40 (RTM: Dioxosilane), separating the working fluid density is 1.108 g/ml, separating the working solution and separating the working fluid density is 1.082 g/ml with 10x phosphate-buffered saline solution 6 ml mother liquor to prepare the solution, separating the working solution 3, separating the working fluid density is 1.042 g/ml, (iv) using centrifugal pipe and paving, separating working solution in 10 ml sterilized centrifugal tube, firstly paving by pipettor 1 Ludox HS40 separating the working liquid 2 mL. then laying 2 # separating working liquid 2 ml Ludox HS-40 (RTM: Dioxosilan e), finally separating the working solution, finishing laying the centrifuge tube and placing on the test tube rack, not shaking, (v) centrifuging, using the workpiece and sterilizing culture dish storing rice seed 1.00 g, placing in 10 ml sterilized centrifugal tube, adding HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution 8 ml, mixing uniformly for 5 minutes on the vortex oscillator, placing for 5 hours at room temperature using supernatant on H1850R and centrifuging 20 minutes, centrifuging using rotor at 4 degrees C at 10000 rotations/minute acceleration value as 5, brake value as 5, centrifuging, removing the supernatant, precipitation using 1 ml HEPES buffer solution suspension to obtain suspension, taking 300 mu l suspension paved on the surface of the solution and paving with a discontinuous density separation working solution Ludox HS40 centrifugal tube, 8 replicates of each sample detection. after paving, on instrument H1850R centrifuge f!
or 15 min, centrifugal condition using rotor at 4 degrees C, 8500 rotations/minute acceleration value as 2, brake value as 0, after forming density grads separating layer, taking out the centrifugal tube. absorbing 1 by pipettor, separating the working solution and separating the working solution of flocculent interface layer solution, (6) using 1 asterisk TE buffer liquid suspension precipitate interface layer the flocculent solution absorbs into the centrifugal tube; adding 500 mu L 10x buffer solution, mixing them uniformly, centrifuging for 3000rpm at 2 min, removing the supernatant removing the Ludox HS-40 (RTM: Dioxosilane), precipitating with 1 Tris-EDTA (TE) buffer solution suspension 1x Tris-EDTA (TE) buffer suspension, (vii) seed bacteria carrying PCR amplification to obtain the 1x TE suspension is total DNA template, performing PCR amplification with upstream primer 799F and downstream primer 1492R, the total volume is 50 ml of PCR reaction system comprises 2 mu l DNA template, 1-5 mu l x Taq reaction, 10 nM/ mu l upstream primer 7 99F 2 mu l, 10 nM/ mu l, downstream primer 1492R 2 mu l, 1U/mu L Taq enzyme 2 mu l, 2.5 M/ mu l dNTPs, 4 mu l double distilled water, 33 mu l reaction program annealing at 94 degrees C for 5 minutes, carrying out denaturation at 52 degrees C for 1 minute and annealing for 1 minute and 72 degrees C extending for 1 minutes after 30 cycles, extending for 10 minutes at 72 degrees C and storing at 4 degrees C, and PCR product size is 750bp, the base sequence of the upstream primer 799F is shown as 5'-aacaggagttagataccctg-3' (SEQ ID NO: 1) and downstream primer 1492R nucleotide sequence and 5'-ggtaccttgttacgactt-3' (SEQ ID NO: 2), (viii) providing 16S rDNA clone library of the amplified PCR product at 1 % agarose gel after electrophoresis, cutting 700-800bp strip under ultraviolet lamp, recovering 750bp strip, using T-vector connection after transforming JM109 competent cells to obtain a transformant, and adding SOC culture medium, where the SOC culture medium containing ampicillin, IPTG and X-Gal lysogeny broth flat plate culture medium, white colony sequencing after colony grows, randomly selecting more than 160, (ix) providing 16S rDNA clone library evaluation according to the formula: C = (1-n1) asterisk 100 %, where n1 is the number of OUT only contains one sequence of N is sequence number of all random total white colony appeared in, and C value calculated using QIIME software, C value is more than 95%, according to the following steps, (x) performing bacterial species analysis using the 16S rDNA sequence of sequencing result in the step (viii) issued on NCBI by sequence comparison to determine bacterial species and by comparison, sequence similarity is more than 97% of the same bacterial species
主权项：1.一种基于硅溶胶密度梯度离心检测种子携带细菌的方法,其特征在于包括以下步 骤： (1)待检水稻种子的准备 取待检水稻种子50粒，先用200mL灭菌水清洗待检水稻种子1～2次后，依次用70％乙醇 2.5mL浸泡2～3min，2.5％的次氯酸钠2.5mL浸泡3～5min，70％乙醇2.5mL浸泡20s～30s，接 着用灭菌水冲洗种子3～4次，将种子晾干后在灭菌研钵中磨碎，用50目筛子过滤，取50目筛 子下面的磨碎水稻种子再用100目筛子过滤，取100目筛子下面的磨碎水稻种子置于直径为 9cm的灭菌培养皿中备用； (2)Ludox HS40等渗母液的配制 用10×PBS缓冲液与Ludox HS40硅溶胶按照1︰9体积比的比率混合制成Ludox HS40等 渗母液，Ludox HS40等渗母液密度为1.308g/mL； (3)配制不连续密度Ludox HS40分离工作液 按如下方法配制1#Ludox HS40分离工作液、2#Ludox HS40分离工作液、3#Ludox HS40 分离工作液； 1#Ludox HS40分离工作液的配制：用10×PBS缓冲液1mL与9mL Ludox HS40等渗母液混 合配制成的溶液即为1#Ludox HS40分离工作液，1#Ludox HS40分离工作液密度为1.108g/ mL； 2#Ludox HS40分离工作液的配制：用10×PBS缓冲液4mL与6mL Ludox HS40等渗母液混 合配制成的溶液即为2#Ludox HS40分离工作液，2#Ludox HS40分离工作液密度为1.082g/ mL； 3#Ludox HS40分离工作液的配制：用10×PBS缓冲液6mL与4mL Ludox HS40等渗母液混 合配制成的溶液即为3#Ludox HS40分离工作液，3#Ludox HS40分离工作液密度为1.042g/ mL； (4)在离心管中铺设不连续密度Ludox HS40分离工作液 在10mL灭菌离心管中，用移液器先铺设1#Ludox HS40分离工作液2mL，然后铺设2# Ludox HS40分离工作液2mL，最后铺设3#Ludox HS40分离工作液2mL，铺设完毕后，将离心管 置于试管架上，不能晃动； (5)离心 ①取步骤(1)在灭菌培养皿中储备的磨碎水稻种子1.00g，置于10mL灭菌离心管中，再 加入1×HEPES缓冲液8mL，在涡旋振荡器上混匀5min，室温放置5h后，取上清液在湘仪 H1850R离心机上离心20min，离心条件为：3号转子，4℃，10000rpm，加速度值设为5，刹车值 设为5； ②离心后，弃上清，沉淀用1×HEPES缓冲液1mL悬浮得悬浮液，取悬浮液300μL铺设于步 骤(4)铺设有不连续密度Ludox HS40分离工作液的离心管中的溶液表面，每个样品检测做8 次重复，铺设好后，在湘仪H1850R离心机上离心15min，离心条件为：3号转子，4℃，8500rpm， 加速度值设为2，刹车值设为0，形成密度梯度分离层后，将离心管取出，用移液器吸取1# Ludox HS40分离工作液与2#Ludox HS40分离工作液之间的絮状界面层溶液； (6)用1×TE缓冲液悬浮沉淀 将吸取的絮状界面层溶液装入新的离心管中，加入500μL 10×PBS缓冲液，混匀后， 3000rpm离心2min，弃上清去除Ludox HS40，沉淀用1×TE缓冲液悬浮得1×TE悬浮液； (7)种子携带细菌PCR扩增 以步骤(6)获得的1×TE悬浮液为总DNA模板，用上游引物799F和下游引物1492R进行 PCR扩增，总体积为50μL的PCR反应体系为：DNA模板2μL，1×Taq reaction 5μL，10nM/μL上 游引物799F 2μL，10nM/μL下游引物1492R 2μL，1U/μL Taq酶2μL，2.5M/μL dNTPs 4μL， ddH2O 33μL；反应程序：94℃预变性5min，从94℃变性1min，52℃退火1min至72℃延伸1min， 30个循环后，72℃延伸10min，4℃保存；PCR产物大小为750bp；所述上游引物799F的碱基序 列如SEQ ID NO：1所示，下游引物1492R的碱基序列如SEQ ID NO：2所示； (8)16S rDNA克隆文库的构建 将扩增得到的PCR产物在1.％的琼脂糖凝胶中电泳，电泳完毕后，在紫外灯下切取700 ～800bp条带，回收750bp条带，用T载体进行连接后转化JM109感受态细胞得到转化子，再加 入SOC培养基培养，将培养后的SOC培养基涂到含有氨苄、IPTG和X-Gal的LB平板培养基上， 待菌落长出后，随机挑取160个以上的白色菌落测序； (9)16S rDNA克隆文库评价 按计算公式：C＝(1-n1/N)×100％，其中n1为只含有一条序列的OTU的数目；N为随机挑 取的所有白色菌落中出现的总的序列数目，并使用QIIME软件进行C值计算，C值为95％以 上，按如下步骤(10)进行细菌种类分析； (10)细菌种类分析 将步骤(8)的测序结果在NCBI上发布的16S rDNA序列进行序列比对确定细菌种类，经 比对，序列相似性为97％以上的归为同一细菌种类。
申请号：CN201610950027.6
公开/公告号：CN106480202A
申请日：2016-10-26
公开/公告日：2017-03-08
申请/专利权人：云南省农业科学院粮食作物研究所
发明/设计人：谷安宇;李小林;徐雨然;叶昌荣;邓伟;汪永华;张锦文
分类号：C12Q1/68;C12Q1/04
主分类号：C12Q1/68
法律状态：实质审查
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