SN T 2059-2008 进出口蜂王浆中链霉素和双氢链霉素残留量测定方法.酶联免疫法.pdf

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1、书书书中华人民共和国出入境检验检疫行业标准犛犖犜进出口蜂王浆中链霉素和双氢链霉素残留量测定方法酶联免疫法犇犲狋犲狉犿犻狀犪狋犻狅狀狅犳狊狋狉犲狆狋狅犿狔犮犻狀犪狀犱犱犻犺狔犱狉狅狊狋狉犲狆狋狅犿狔犮犻狀狉犲狊犻犱狌犲狊犻狀狉狅狔犪犾犼犲犾犾狔犳狅狉犻犿狆狅狉狋犪狀犱犲狓狆狅狉狋犈狀狕狔犿犲犾犻狀犽犲犱犻犿犿狌狀狅狊狅狉犫犲狀狋犪狊狊犪狔发布实施中华人民共和国国家质量监督检验检疫总局发布书书书前G21G21言本标准的附录G21为资料性附录G21本标准由国家认证认可监督管理委员会提出并归口G21本标准起草单位G22中华人民共和国浙江出入境检验检疫局G21本标准主要起草人G22施伟良G23张晓峰G2

2、3朱振江G23程洁G23苏宗仁G21本标准系首次发布的出入境检验检疫行业标准G21G21G21G22G21G23G21G22G23G24G22G21G22G22G进出口蜂王浆中链霉素和双氢链霉素残留量测定方法G21酶联免疫法G26G21范围本标准规定了蜂王浆和王浆冻干粉中链霉素和双氢链霉素残留量的酶联免疫测定方法G21本标准适用于蜂王浆和王浆冻干粉中链霉素和双氢链霉素残留总量的测定G21G21G21规范性引用文件下列文件中的条款通过本标准的引用而成为本标准的条款G21凡是注明日期的引用文件G24其随后所有的修改单G25不包括勘误的内容G26或修订版均不适用于本标准G24然而G24鼓励根据本标准

3、达成协议的各方研究是否可使用这些文件的最新版本G21凡是不注明日期的引用文件G24其最新版本适用于本标准G21G22G23G27G24G25G25G26G27G21分析实验室用水规格和试验方法G25G22G23G27G24G25G25G26G27G28G28G29G29G27G24G2AG2BG2CG2DG25G29G25G22G28G29G26G2EG26G27G21方法提要本标准以酸性缓冲液来沉淀蜂王浆中蛋白质G23提取残留的链霉素和双氢链霉素G24然后以G2FG30G23柱净化G21处理后样品中残留的链霉素和双氢链霉素与酶标记链霉素共同竞争结合链霉素抗体G24同时链霉素抗体结合至包被有绵

4、羊抗兔G31G32G22的抗体的微孔板上G24通过洗涤除去未结合的链霉素和双氢链霉素和酶标记链霉素G24然后加入底物显色G24用酶标仪测定吸光度G24根据吸光度值得出蜂王浆中链霉素和双氢链霉素的含量G21G28G21试剂和材料除去注明外G24所有试剂均为分析纯G24水为G22G23G27G24G25G25G26G27规定的一级水G21G28G33G26G21链霉素检测试剂盒G25参见附录G21G26G21G28G33G21G21甲醇G21G28G33G27G21G34G35G23缓冲溶液G22称取G28G33G28G36G32磷酸氢二钠G24G37G33G27G32磷酸二氢钾G24G37G33

5、G27G32氯化钾G24G2DG37G32氯化钠G24G37G33G36G38G30吐温G39G26G37G24用水定容至G28G37G37G37G38G30G24用磷酸G27氢氧化钠调节G3AG2F至G2EG33G36G21G28G33G28G21庚烷磺酸钠缓冲液G22称取G28G37G33G28G32庚烷磺酸钠G29G3BG2FG2DG25G3BG2FG27G26G25G34G3CG2DG3DG3EG2AG24G28G28G33G3FG32磷酸钠G25G3DG3EG2DG40G3CG3FG2BG28G27G2FG27G3CG26用水溶解并定容至G28G37G37G37G38G30G24用磷

6、酸调节G3AG2F至G27G33G37G21G28G33G23G21G28G37G41甲醇G22量取G28G37G38G30甲醇G25G3FG33G27G26用水定容至G28G37G37G38G30G21G28G33G29G21G2FG30G23小柱G22G3CG3EG42G43G42G25或相当产品G26G24G2DG38G30G25G25G37G38G32G26G21G28G33G2AG21链霉素和双氢链霉素标准品G22纯度大于等于G29G26G41G21G28G33G25G21链霉素和双氢链霉素标准品溶液的配制G22称取G37G33G27G36G32链霉素或双氢链霉素G24用甲醇定容至G

7、28G37G38G30G24配制成G27G36G38G32G27G38G30的储存液G24于G44G27G37G45条件下保存G21G23G21仪器G23G33G26G21酶标仪G21G23G33G21G21G26道移液器G22G28G37G22G30G23G28G37G37G22G30G21G23G33G27G21单道移液器G22G28G37G22G30G23G28G37G37G22G30G24G27G37G22G30G23G27G37G37G22G30G24G28G37G37G22G30G23G28G37G37G37G22G30和G27G38G30G23G28G37G38G30G21G23

8、G33G28G21混合振荡器G21G28G21G22G21G23G21G22G23G24G22G21G22G22GG23G33G23G21高速低温离心机G22G25G37G37G37G46G27G38G43G2AG21G23G33G29G21固相萃取装置G21G23G33G2AG21氮吹仪G21G23G33G25G21具塞试管G22G36G37G38G30G21G23G33G24G21电子天平G22G37G33G37G28G32G23G28G37G37G32G21G23G33G26G22G21酸度计G21G29G21试样的制备和保存G29G33G26G21试样的制备原始样品总量不得少于G27G

9、37G37G32G24蜂王浆充分搅拌均匀后G24将样品分成两等份G2C冻干粉采用四分法G24将样品分成两等份G21分好的样品装入洁净容器G24加封并做标识G21G29G33G21G21试样的保存试样放置G44G27G37G45G23G44G28G26G45条件下保存G21G2AG21分析步骤G2AG33G26G21提取称取G27G33G37G32蜂王浆样品G24置于G36G37G38G30具塞试管中G24加入G26G38G30庚烷磺酸钠缓冲液G25G3FG33G3FG26G24充分混匀G24G28G36G45条件下G25G37G37G37G46G27G38G43G2A离心G28G37G38G4

10、3G2A直至清亮G24取上层液备用G21G2FG30G23小柱G25G3FG33G25G26G24依次用G28G38G30甲醇G25G3FG33G27G26和G28G38G30水活化G24吸取G28G33G36G38G30样品提取液上柱G24然后G2DG38G30水洗G24G28G38G30G28G37G41甲醇G25G3FG33G36G26洗柱G24去除残留的液体G24氮气吹G27G38G43G2A干燥柱子G21然后以G27G38G30甲醇G25G3FG33G27G26洗脱G24将样品收集于干净塑料试管G24氮气吹干G21以G2DG38G30G34G35G23缓冲液G25G3FG33G2DG

11、26溶解吹干残留物G24用于G47G30G31G34G21检测G21最后样品稀释倍数为G28G37G21王浆冻干粉则用水以G28G48G27比例稀释G24充分浸泡G25G27G49以上G26后G24称取G27G33G37G32按照上述王浆前处理方法进行提取G24最后以G27G38G30G34G35G23缓冲液溶解吹干残留物G24最后样品稀释倍数为G27G37G21G2AG33G21G21测定条件G26G26G2AG33G21G33G26G21操作条件所有操作应在室温下G25G27G37G45G23G27G3FG45G26进行G24链霉素试剂盒中所有试剂的温度均应回升至室温G25G27G37G4

12、5G23G27G3FG45G26后方可使用G21G2AG33G21G33G21G21洗板条件人工洗涤次数G36次以上G24每次加入洗涤液量为G27G36G37G22G30G2C自动洗板可以预定G36次G21G2AG33G21G33G27G21酶标仪测定条件酶标仪测定波长为G3FG36G37G2AG38G21G2AG33G27G21测定步骤G2AG33G27G33G26G21将测定需用的微孔板备齐并插入微孔架上G24记录标准品及样品等在微孔架上的位置G21G2AG33G27G33G21G21吸取G28G37G37G22G30零浓度标准品于孔G21G28G23G21G27G2C并吸取G36G37G

13、22G30零浓度标准品于孔G23G28G23G23G27G2C分别吸取G36G37G22G30链霉素标准溶液G25浓度分别为G22G37G33G27G36G23G37G33G36G23G28G33G37G23G27G33G37G23G28G37G33G37G23G27G37G33G37G2AG32G27G38G30G26于孔G3BG28G23G3BG27G28G2FG28G23G2FG27G2C分别吸取G36G37G22G30样品提取液于其余微孔中G21测定中吸取不同的试剂和样品溶液时应更换吸头G21G2AG33G27G33G27G21分别吸取G27G36G22G30链霉素酶标记物溶液于除G2

14、1G28G23G21G27外的每一个微孔G21G2AG33G27G33G28G21分别吸取G27G36G22G30链霉素抗体溶液于除G21G28G23G21G27外的每一个微孔G21G2AG33G27G33G23G21用封口膜封孔条G24并持微孔板在台面上以圆周运动方式混匀G21G28G26G21给出该信息是为了方便本标准的使用者G24并不表示对某一产品操作步骤的认可G21如果其他产品的操作步骤有不同G24需经实验评估后采用G21G2AG33G27G33G29G21将酶标板置于G3FG45避光温育G28G49G21G27G21G22G21G23G21G22G23G24G22G21G22G22G

15、G2AG33G27G33G2AG21倒出孔中的液体G24将微孔架反扣在吸水纸上反复拍打G24以除去孔中过多的残液G24但不能使微孔干燥G21然后立即用洗涤缓冲液按G2EG33G27G33G27条件进行洗板G21要注意不能使微孔干燥G21G2AG33G27G33G25G21迅速加入G28G37G37G22G30底物溶液于每一个微孔底部G24然后持微孔板在台面上以圆周运动方式混匀后G24于G27G37G45G23G27G3FG45避光温育G2DG37G38G43G2AG21G2AG33G27G33G24G21加入G28G37G37G22G30反应终止液于每一个微孔G24然后持微孔板在台面上以圆周运

16、动方式混匀后G24将微孔架置于酶标仪中G24在G3FG36G37G2AG38处测量吸光度G25应在加入反应终止液后G2DG37G38G43G2A内读取吸光度G26G21G2AG33G28G21平行试验按以上步骤G24对同一标准溶液G23同一样品溶液均应进行平行试验测定G21G2AG33G23G21空白试验除不加入试样外G24均按上述步骤进行G21G2AG33G29G21阳性质控每次测定均应做一个添加链霉素和双氢链霉素标准品溶液G25G3FG33G26G26的监控样品测定G24以确定实验过程的操作准确性G21G25G21结果计算从标准品和样品的吸光度G25G3CG35G26值中G24减去空白孔G

17、21G28G23G21G27的平均G3CG35值G21标准品和样品的G3CG35平均值除以零标准G25G23G28G23G23G27G26的平均G3CG35值G24再乘以G28G37G37G21零标准为G28G37G37G41G25最大百分比吸光度值G26G24其他G3CG35值为最大吸光度值的百分数G21以吸光度的百分比值G25G21G27G21G37G26为纵坐标G25G41G26G24链霉素标准溶液浓度G25G2AG32G27G38G30G26的对数值为横坐标G24绘制标准工作曲线G21从标准工作曲线上得到试样中相应的链霉素浓度后G24结果按式G25G28G26进行计算G22G22G23

18、G24G25G26G25G28G37G37G37G27G25G28G37G37G37G2DG2DG2DG2DG2DG2DG2DG2DG2DG2DG2DG25G28G26G21G21式中G22G22G28G28G28样品中链霉素和双氢链霉素的残留总量G24单位为微克每千克G25G22G32G27G4AG32G26G2CG24G28G28G28从标准工作曲线上得到的样品中链霉素和双氢链霉素浓度G24单位为纳克每毫升G25G2AG32G27G38G30G26G2CG26G28G28G28样品溶液的最终定容体积G24单位为毫升G25G38G30G26G2CG27G28G28G28样品溶液所代表的最终试

19、样质量G24单位为克G25G32G26G21也可以用各种酶标仪的数据处理软件进行计算G21所得结果表示至一位小数G21G24G21确证试验如被测样品中为阳性结果时G24应用其他方法进行确证G21G26G22G21本方法测定低限和回收率G26G22G33G26G21方法测定低限G28G37G22G32G27G4AG32G21G26G22G33G21G21回收率本方法中链霉素和双氢链霉素添加浓度及回收率的试验数据G22G3EG26G21链霉素G28G28G28添加量为G28G37G22G32G27G4AG32时G24回收率为G26G29G41G23G28G28G3FG41G2CG28G28G28添

20、加量为G27G37G22G32G27G4AG32时G24回收率为G26G2DG33G36G41G23G28G37G29G33G36G41G2CG28G28G28添加量为G27G37G37G22G32G27G4AG32时G24回收率为G26G25G33G3FG41G23G28G37G28G33G25G41G2CG4BG26G21双氢链霉素G28G28G28添加量为G28G37G22G32G27G4AG32时G24回收率为G2EG29G41G23G28G37G26G41G2CG28G28G28添加量为G27G37G22G32G27G4AG32时G24回收率为G26G27G41G23G28G37G2

21、EG33G36G41G2CG28G28G28添加量为G27G37G37G22G32G27G4AG32时G24回收率为G2EG29G33G27G41G23G28G37G27G33G26G41G21G2DG21G22G21G23G21G22G23G24G22G21G22G22G附G21录G21G24G25资料性附录G26链霉素试剂盒G25荷兰G25G26G27G28G29G2AG2BG24G2CG22G28G21G23G2BG2DG24公司产品G26G21G26G24G33G26G21试剂盒组成G24G33G26G33G26G21预包被抗体的G29G25孔板G22G28G27条G4CG26孔G21

22、G24G33G26G33G21G21链霉素标准溶液G22G37G23G37G33G27G36G23G37G33G36G23G28G33G37G23G27G33G37G23G28G37G33G37G23G27G37G33G37和G28G37G37G2AG32G27G38G30G21G24G33G26G33G27G21链霉素酶标记物冻干粉G22根据链霉素试剂盒中说明G24可用稀释缓冲液配制成链霉素酶标记物溶液G21G24G33G26G33G28G21抗链霉素抗体冻干粉G22根据链霉素试剂盒中说明G24可用稀释缓冲液配制成抗链霉素抗体溶液G21G24G33G26G33G23G21底物G24G4DG2

23、3溶液G21G24G33G26G33G29G21稀释缓冲液G21G24G33G26G33G2AG21洗涤浓缩液G22可用水G28G37倍稀释后使用G21G24G33G26G33G25G21反应终止液G21试剂盒应在G3FG45G23G26G45避光条件下保存G24溶解后的酶标记物和抗体溶液需G44G28G36G45条件冻存G21G24G33G21G21标准校准曲线标准校准曲线见图G21G33G28G21G28G4EG44G28G2EG4FG28G27G25G50G2AG29G51G25G36G4FG29G36G2AG27G4EG37G4FG29G29G36G3F图G24G33G26G21链霉素

24、标准曲线G27G26G21给出该信息是为了方便本标准的使用者G24并不表示对某一产品的认可G21如果其他产品具有相同的效果G24需经实验评估后使用这些等效产品G21G3FG21G22G21G23G21G22G23G24G22G21G22G22GG21G22G23G24G25G22G23G26G27G28G28G24G29G27G22G2AG2BG2CG2DG2EG2EG2BG2FG28G26G2FG23G26G2FG23G24G2DG28G2AG22G23G30G2FG2BG2DG31G24G2FG28G28G24G29G2EG33G32G2CG2DG2EG2EG2BG2FG28G26G2FG

25、23G26G25G2FG2EG33G23G22G33G22G2EG24G26G34G35G36G24G23G2BG2DG2AG2DG37G2FG2BG2DG22G28G2FG28G26G27G37G37G23G24G26G2DG2BG2FG2BG2DG22G28G27G26G30G2DG28G2DG2EG2BG23G2FG2BG2DG22G28G22G2AG2BG2CG24G38G24G22G33G39G24G2EG2EG3AG24G33G3BG34G39G2DG37G22G2AG36G2CG2DG28G2FG33G32G2CG2DG2EG2EG2BG2FG28G26G2FG23G26G25G

26、2FG2EG30G2FG2DG28G39G35G26G23G2FG2AG2BG24G26G34G35G3CG2CG24G3DG2DG2FG28G3EG3FG28G2BG23G35G40G3FG29G2DG2BG41G28G2EG33G24G37G2BG2DG22G28G2FG28G26G42G3BG2FG23G2FG28G2BG2DG28G24G43G3BG23G24G2FG3BG22G2AG2BG2CG24G38G24G22G33G39G24G2EG2EG3AG24G33G3BG34G39G2DG37G22G2AG36G2CG2DG28G2FG33G32G2CG2DG2EG2EG2BG2FG

27、28G26G2FG23G26G25G2FG2EG30G2FG2DG28G39G35G26G2FG2AG2BG24G26G34G35G44G24G2DG40G39G2DG2FG28G3EG45G2CG2DG24G21G24G28G3EG40G29G2DG2FG22G3CG2CG2FG28G3EG24G3CG2CG24G28G40G3DG2DG2FG28G3EG3CG2CG3BG24G46G2DG24G36G2CG24G28G3EG24G3CG2CG22G28G3EG40G23G24G28G45G3BG32G2CG2DG2EG2EG2BG2FG28G26G2FG23G26G2DG2EG33G23G

28、22G2AG24G2EG2EG2DG22G28G2FG39G2EG2BG2FG28G26G2FG23G26G22G2AG3FG28G2BG23G35G40G3FG29G2DG2BG2DG28G2EG33G24G37G2BG2DG22G28G2FG28G26G47G3BG2FG23G2FG28G2BG2DG28G24G33G23G22G30G3BG39G3EG2FG2BG24G26G2AG22G23G2BG2CG24G2AG2DG23G2EG2BG2BG2DG30G24G33G36G21G22G21G23G21G22G23G24G22G21G22G22GG48G24G2BG24G23G30G2D

29、G28G2FG2BG2DG22G28G22G2AG2EG2BG23G24G33G2BG22G30G35G37G2DG28G2FG28G26G26G2DG2CG35G26G23G22G2EG2BG23G24G33G2BG22G30G35G37G2DG28G23G24G2EG2DG26G3BG24G2EG2DG28G23G22G35G2FG39G3DG24G39G39G35G2AG22G23G2DG30G33G22G23G2BG2FG28G26G24G29G33G22G23G2BG28G3FG28G49G35G30G24G40G39G2DG28G4AG24G26G41G30G30G3BG28G22

30、G2EG22G23G34G24G28G2BG2FG2EG2EG2FG35G4BG21G45G37G22G33G24G32G2CG2DG2EG2EG2BG2FG28G26G2FG23G26G2EG33G24G37G2DG2AG2DG24G2EG2BG2CG24G30G24G2BG2CG22G26G2EG22G2AG26G24G2BG24G23G30G2DG28G2FG2BG2DG22G28G34G35G3FG4CG41G45G27G30G24G2BG2CG22G26G22G2AG2EG2BG23G24G33G2BG22G30G35G37G2DG28G2FG28G26G26G2DG2CG35G26

31、G23G22G40G2EG2BG23G24G33G2BG22G30G35G37G2DG28G23G24G2EG2DG26G3BG24G2EG2DG28G23G22G35G2FG39G3DG24G39G39G35G2FG28G26G23G22G35G2FG39G3DG24G39G39G35G33G22G25G26G24G23G33G32G2CG2DG2EG2EG2BG2FG28G26G2FG23G26G2DG2EG2FG33G33G39G2DG37G2FG34G39G24G2BG22G2BG2CG24G2EG37G23G24G24G28G26G24G2BG24G23G30G2DG28G2FG2B

32、G2DG22G28G22G2AG2EG2BG23G24G33G2BG22G30G35G37G2DG28G2FG28G26G26G2DG2CG35G26G23G22G2EG2BG23G24G33G2BG22G30G35G37G2DG28G23G24G2EG40G2DG26G3BG24G2EG2DG28G23G22G35G2FG39G3DG24G39G39G35G2FG28G26G23G22G35G2FG39G3DG24G39G39G35G33G22G25G26G24G23G33G4DG21G4EG22G23G30G2FG2BG2DG31G24G23G24G2AG24G23G24G28G37G24

33、G2EG32G2CG24G2AG22G39G39G22G25G2DG28G3EG28G22G23G30G2FG2BG2DG31G24G26G22G37G3BG30G24G28G2BG2EG37G22G28G2BG2FG2DG28G33G23G22G31G2DG2EG2DG22G28G2EG25G2CG2DG37G2CG24G2BG2CG23G22G3BG3EG2CG23G24G2AG24G23G24G28G37G24G2DG28G2BG2CG2DG2EG2BG24G29G2BG24G37G22G28G40G2EG2BG2DG2BG3BG2BG24G33G23G22G31G2DG2EG2DG22

34、G28G2EG22G2AG2BG2CG2DG2EG2EG2BG2FG28G26G2FG23G26G33G21G22G23G26G2FG2BG24G26G23G24G2AG24G23G24G28G37G24G2EG24G2EG3BG34G2EG24G47G3BG24G28G2BG2FG30G24G28G26G30G24G28G2BG2EG2BG22G24G22G23G23G24G31G2DG2EG2DG22G28G2EG22G2AG24G2FG28G35G22G2AG2BG2CG24G2EG24G33G3BG34G39G2DG37G2FG2BG2DG22G28G2EG26G22G28G22G2B

35、G2FG33G33G39G35G33G4FG22G25G24G31G24G23G33G2FG23G2BG2DG24G2EG2BG22G2FG3EG23G24G24G30G24G28G2BG2EG34G2FG2EG24G26G22G28G2BG2CG2DG2EG2EG2BG2FG28G26G2FG23G26G2FG23G24G24G28G37G22G3BG23G2FG3EG24G26G2BG22G2DG28G31G24G2EG2BG2DG3EG2FG2BG24G2BG2CG24G33G22G2EG2EG2DG34G2DG39G2DG2BG35G22G2AG2FG33G33G39G35G2DG28

36、G3EG2BG2CG24G30G22G2EG2BG23G24G37G24G28G2BG24G26G2DG2BG2DG22G28G2EG22G2AG2BG2CG24G28G22G23G30G2FG2BG2DG31G24G26G22G37G3BG30G24G28G2BG2EG2DG28G26G2DG37G2FG2BG24G26G34G24G39G22G25G33G21G22G23G3BG28G26G2FG2BG24G26G23G24G2AG24G23G24G28G37G24G2EG24G2BG2CG24G39G2FG2BG24G2EG2BG24G26G2DG2BG2DG22G28G22G2AG2B

37、G2CG24G28G22G23G30G2FG2BG2DG31G24G26G22G37G3BG30G24G28G2BG23G24G2AG24G23G23G24G26G2BG22G2FG33G33G39G2DG24G2EG33G50G43G27G32G51G51G52G4DG44G2FG2BG24G23G2AG22G23G2FG28G2FG39G35G2BG2DG37G2FG39G39G2FG34G22G23G2FG2BG22G23G35G3BG2EG24G28G45G33G24G37G2DG2AG2DG37G2FG2BG2DG22G28G2FG28G26G2BG24G2EG2BG30G24G2B

38、G2CG22G26G2EG33G25G50G43G27G32G51G51G52G4DG28G4BG53G53G4DG24G28G24G47G41G45G54G55G51G53G51G22G4BG53G52G56G26G55G21G38G23G2DG28G37G2DG33G39G24G32G2CG24G30G2DG37G23G22G2BG2DG2BG23G24G34G2FG2EG24G26G3FG41G27G4AG2DG2BG37G22G28G2EG2DG2EG2BG2EG22G2AG4BG4DG2EG2BG23G2DG33G2EG24G24G2FG37G2CG52G25G24G39G39G2E

39、G24G33G23G24G37G22G2FG2BG24G26G25G2DG2BG2CG2EG2CG24G24G33G2FG28G2BG2DG34G22G26G2DG24G2EG2BG22G23G2FG34G34G2DG2BG41G3EG50G33G27G2EG33G24G37G2DG2AG2DG37G2FG28G2BG2DG34G22G26G35G25G23G2FG34G34G2DG2BG2FG28G2BG2DG40G45G2BG23G24G33G2BG22G30G35G37G2DG28G26G24G24G28G49G35G30G24G39G2FG34G24G39G39G24G26G45G2B

40、G23G24G33G2BG22G30G35G37G2DG28G25G24G28G49G35G30G24G37G22G28G3DG3BG3EG2FG2BG24G26G2FG28G26G45G2BG23G24G33G2BG22G30G35G37G2DG28G2EG2BG2FG28G26G2FG23G26G22G23G2EG2FG30G33G39G24G2FG23G24G2FG26G26G24G26G2BG22G2BG2CG24G33G23G24G37G22G2FG2BG24G26G25G24G39G39G2EG2AG22G39G39G22G25G24G26G34G35G2FG2EG2DG28G40

41、G3EG39G24G2DG28G37G3BG34G2FG2BG2DG22G28G2EG2BG24G33G33G32G2CG24G2EG33G24G37G2DG2AG2DG37G2FG28G2BG2DG34G22G26G2DG24G2EG2FG23G24G34G22G3BG28G26G34G35G2BG2CG24G2DG30G30G22G34G2DG39G2DG2EG24G26G2FG28G2BG2DG34G22G26G2DG24G2EG2FG28G26G2FG2BG2BG2CG24G2EG2FG30G24G2BG2DG30G24G2AG23G24G24G45G2BG23G24G33G2BG22

42、G30G35G37G2DG28G27G48G2DG2CG35G26G23G22G2EG2BG23G24G33G2BG22G30G35G37G2DG28G25G33G23G24G2EG24G28G2BG2DG28G2BG2CG24G2EG2BG2FG28G26G2FG23G26G2EG22G39G3BG2BG2DG22G28G22G23G2EG2FG30G33G39G24G26G2FG28G26G24G28G40G49G35G30G24G37G22G28G3DG3BG3EG2FG2BG24G26G45G2BG23G24G33G2BG22G30G35G37G2DG28G37G22G30G33G24

43、G2BG24G2AG22G23G2BG2CG24G45G2BG23G24G33G2BG22G30G35G37G2DG28G2FG28G2BG2DG34G22G26G35G34G2DG28G26G2DG28G3EG2EG2DG2BG24G2FG25G37G22G30G33G24G2BG2DG2BG2DG31G24G24G28G49G35G30G24G2DG30G30G3BG28G22G2FG2EG2EG2FG35G26G33G27G2AG2BG24G23G2FG28G2DG28G37G3BG34G2FG2BG2DG22G28G2BG2DG30G24G22G2AG4BG2CG24G2BG2CG24

44、G28G22G28G40G34G22G3BG28G26G25G24G28G49G35G30G24G39G2FG34G24G39G39G24G26G26G23G24G2FG3EG24G28G2BG2EG2FG23G24G23G24G30G22G31G24G26G2DG28G2FG25G2FG2EG2CG2DG28G3EG2EG2BG24G33G33G32G2CG24G2FG30G22G3BG28G2BG22G2AG45G2BG23G24G33G2BG22G30G35G37G2DG28G24G28G49G35G30G24G37G22G28G3DG3BG3EG2FG2BG24G2DG2EG31G2D

45、G2EG3BG2FG39G2DG2EG24G26G34G35G2BG2CG24G2FG26G26G2DG2BG2DG22G28G22G2AG2FG37G2CG23G22G30G22G3EG24G28G2EG3BG34G2EG2BG23G2FG2BG24G25G2BG24G2BG23G2FG30G24G2BG2CG35G39G34G24G28G49G2DG26G2DG28G24G24G32G57G43G26G33G43G22G3BG28G26G24G28G49G35G30G24G37G22G28G3DG3BG3EG2FG2BG24G2BG23G2FG28G2EG40G2AG22G23G30G2E

46、G2BG2CG24G37G22G39G22G23G39G24G2EG2EG37G2CG23G22G30G22G3EG24G28G2DG28G2BG22G2FG37G22G39G22G3BG23G24G26G33G23G22G26G3BG37G2BG33G32G2CG24G2EG3BG34G2EG2BG23G2FG2BG24G23G24G2FG37G2BG2DG22G28G2DG2EG2EG2BG22G33G33G24G26G34G35G33G2CG22G2BG22G40G30G24G2BG23G2DG37G2FG39G39G35G2FG2BG58G59G5AG28G30G33G32G2CG24

47、G22G33G2BG2DG37G2FG39G26G24G28G2EG2DG2BG35G2DG2EG2DG28G31G24G23G2EG24G39G35G33G23G22G33G22G23G2BG2DG22G28G2FG39G2BG22G2BG2CG24G45G2BG23G24G33G2BG22G30G35G37G2DG28G27G48G2DG2CG35G26G23G22G40G2EG2BG23G24G33G2BG22G30G35G37G2DG28G37G22G28G37G24G28G2BG23G2FG2BG2DG22G28G2DG28G2BG2CG24G2EG2FG30G33G39G24G33

48、G25G21G22G21G23G21G22G23G24G22G21G22G22GG58G21G3AG24G2FG3EG24G28G2BG2EG2FG28G26G30G2FG2BG24G23G2DG2FG39G2EG5BG28G39G24G2EG2EG22G2BG2CG24G23G25G2DG2EG24G2EG33G24G37G2DG2AG2DG24G26G24G2FG39G39G2BG2CG24G37G2CG24G30G2DG37G2FG39G23G24G2FG3EG24G28G2BG2EG2EG2CG22G3BG39G26G34G24G27G33G3AG33G3EG23G2FG26G24G3

49、3G2FG25G2FG2BG24G23G30G2DG2EG2BG2CG24G2AG2DG23G2EG2BG3EG23G2FG26G24G25G2FG2BG24G23G33G23G24G2EG37G23G2DG34G24G26G34G35G50G43G27G32G51G51G52G4DG33G58G33G4BG21G3FG28G49G35G30G24G2DG30G30G3BG28G22G2FG2EG2EG2FG35G4AG2DG2BG2AG22G23G2BG2CG24G47G3BG2FG28G2BG2DG2BG2FG2BG2DG31G24G2FG28G2FG39G35G2EG2DG2EG22G2AG2EG2BG23G24G33G2BG22G30G35G37G2DG28G33G25G2EG24G24G2FG28G28G24G29G27G26G58G33G4DG21G57G24G2BG2CG2FG28G22G39G