CN1502983A - 测定昆虫对杀虫剂抗性的方法和试剂盒 - Google Patents

测定昆虫对杀虫剂抗性的方法和试剂盒 Download PDF

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CN1502983A
CN1502983A CNA021589313A CN02158931A CN1502983A CN 1502983 A CN1502983 A CN 1502983A CN A021589313 A CNA021589313 A CN A021589313A CN 02158931 A CN02158931 A CN 02158931A CN 1502983 A CN1502983 A CN 1502983A
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kit
damping fluid
hydrogen peroxide
tetramethyl benzidine
solvent
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CN100498302C (zh
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李・汉・林
李·汉·林
・瓦西・艾哈迈德
纳兹尼·瓦西·艾哈迈德
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MEDICINE RESEARCH ACADEMY
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Abstract

使用单个昆虫测定复合功能氧化酶活性提高的方法，其中包含用杵在酸性pH缓冲液中将单个的昆虫匀浆，优选在如Eppendorf试管的小试管中研磨，加入四甲基对二氨基联苯缓冲液作为基质，加入过氧化氢作为氧化剂，并检查蓝色度，其强度表示复合功能氧化酶活性提高的水平。本发明还提供了在本方法中使用的试剂盒，其包含单独的下述容器：I.一种酸性的pH缓冲液，II. 四甲基对二氨基联苯，III.一种溶解四甲基对二氨基联苯的溶剂，和IV.过氧化氢。

Description

测定昆虫对杀虫剂抗性的方法和试剂盒
技术领域
本发明涉及测定昆虫对杀虫剂抗性的方法和试剂盒，更具体地提供了用来测定昆虫对拟除虫菊酯类杀虫剂抗性的田间使用的方法和试剂盒。
背景技术
化学杀虫剂目前仍然是防治如疟疾、登革热、丝虫病等媒介传播疾病的最重要的试剂。自从1940年发明第一种合成杀虫剂DDT以来，上述疾病的传播造成无数的人死亡的现象大大减少了。可是，持续地使用杀虫剂导致携带上述疾病，主要是昆虫媒介的抗性的发展，昆虫的抗药性降低了对媒介以及它们传播的疾病的防治效果。抗性的测定主要是基于世界卫生组织(WHO)的标准试验方法，这些方法对使用者来讲是特别不易使用的，使用试剂盒的货架寿命短并需要熟练的人员进行试验并解释试验结果。结果，人们一般都不知道许多种昆虫媒介的敏感性水平，而昆虫的敏感性影响着防治的结果。
在防治疾病传播媒介中的一个重大突破应该是开发快速田间试剂盒。
通常，抗性的生化基础是下述3种机理：
1)以有机磷和氨基甲酸酯为靶标的非特定酯酶水平的增加；
2)复合功能的氧化酶对拟除虫菊酯的活性提高；和
3)乙酰胆碱酯酶对有机磷和氨基甲酸酯的非敏感性。
发明内容
本发明提供了使用单个昆虫测定昆虫中复合功能氧化酶活性提高的方法和试剂盒，本方法和试剂盒可快速和简便的使用，且获得的结果非常容易解释。
根据本发明的试剂盒包含单独的下述容器：
I.一种酸性的pH缓冲液，
II.四甲基对二氨基联苯，
III.一种溶解四甲基对二氨基联苯的溶剂，和
IV.过氧化氢。
用于田间的试剂盒还包括进行试验的设备，例如混合容器、如Eppendorf试管的试验试管、移液管和微检测盘(micro-assay plate)，通常还有颜色图谱。
缓冲液的pH优选为4-6，更优选约5。
pH缓冲液优选为乙酸钠。
溶剂优选为无水甲醇。
优选使用的过氧化氢是3％w/w的水溶液。
本发明的方法包含用杵在缓冲液中将单个的昆虫匀浆，优选在如Eppendorf试管的小试管中研磨，加入四甲基对二氨基联苯缓冲液作为基质，加入过氧化氢作为氧化剂，并检查蓝色度，其强度表示复合功能氧化酶活性提高的水平，且由此确定对拟除虫菊酯杀虫剂的抗性。
非抗性昆虫的着色非常浅，其随着抗性水平的增加变为深蓝。
通过与颜色图谱比较，可用肉眼评价着色度，如果需要更准确的结果，可在630nm用免疫-测定读数器扫描。
本发明的优点是最先获得了仅需要对单个的昆虫进行测定，就可测定复合功能氧化酶活性提高的试剂盒，使用本方法可以非常及时地获得快速试验结果，且可以不需要仪器，通过目测和比色计读数。本试剂盒和方法花费的人力少且不需要特殊训练即可使用。而且，本试剂盒具有较长货架寿命，可在室温下稳定数周。
应用本试剂盒可以
1.相当简单地测定抗性，
2.确保用于防治害虫和疾病媒介的化学杀虫剂是有效的，从而减少死亡率、医疗费用和人力，
3.能够稳定和定期地监测靶标昆虫的敏感性，和
4.对新防治药剂、防治策略和理解杀虫剂作用模式的设计进行支持。
具体实施方式
分别制备包含下列物质的5个瓶：10ml乙酸钠缓冲液(pH5)，2.5ml无水甲醇，5mg四甲基对二氨基联苯，7.5ml乙酸钠缓冲液(pH5)和5ml过氧化氢(3％)。
为了进行试验，将无水甲醇倒入四甲基对二氨基联苯中并振荡使四甲基对二氨基联苯完全溶解，且所得溶液用7.5ml乙酸钠缓冲液缓冲。用杵在Eppendorf试管中的一滴缓冲液中将一只蚊子匀浆，然后用缓冲液稀释到Eppendorf试管的1ml标记。
将两滴匀浆液滴入到微检测盘的每个孔中，然后滴入4滴四甲基对二氨基联苯溶液和一滴过氧化氢。
然后肉眼计分评价结果，通过与色图标准比较检查颜色或在630nm用免疫测定读数器扫描。

Claims (16)

1.一种测定昆虫中复合功能氧化酶活性提高的方法，包含在酸性pH缓冲液中将单个的昆虫匀浆，加入酸性pH缓冲的四甲基对二氨基联苯溶液作为基质，加入过氧化氢作为氧化剂，并检查蓝色度，所述蓝色度的强度表示复合功能氧化酶活性提高的水平。
2.根据权利要求1所述的方法，其特征在于，所述的方法使用了试剂盒，该试剂盒包含单独的下述容器：
I.一种酸性的pH缓冲液，
II.四甲基对二氨基联苯，
III.一种溶解四甲基对二氨基联苯的溶剂，和
IV.过氧化氢。
3.根据权利要求1或2所述的方法，其特征在于，所述的缓冲液是pH4-6的缓冲液。
4.根据权利要求3所述的方法，其特征在于，所述的缓冲液是pH5的缓冲液。
5.根据权利要求1或2所述的方法，其特征在于，所述的缓冲液是乙酸钠。
6.根据权利要求1-5任意一项所述的方法，其特征在于，所述的溶剂是甲醇。
7.根据权利要求1-6任意一项所述的方法，其特征在于，所述的过氧化氢是3％w/w的水溶液。
8.权利要求1-7任意一项所述的方法中使用的试剂盒，包含单独的下述容器：
I.一种酸性的pH缓冲液，
II.四甲基对二氨基联苯，
III.一种溶解四甲基对二氨基联苯的溶剂，和
IV.过氧化氢。
9.根据权利要求8所述的试剂盒，其特征在于，所述的缓冲液是pH4-6的缓冲液。
10.根据权利要求9所述的试剂盒，其特征在于，所述的缓冲液是pH5的缓冲液。
11.根据权利要求8所述的试剂盒，其特征在于，所述的缓冲液是乙酸钠。
12.根据权利要求8-11任意一项所述的试剂盒，其特征在于，所述的溶剂是甲醇。
13.根据权利要求8-12任意一项所述的试剂盒，其特征在于，所述的过氧化氢是3％w/w的水溶液。
14.根据权利要求8-13任意一项所述的试剂盒，其特征在于，所述的试剂盒还包含进行试验的设备。
15.根据权利要求14所述的试剂盒，其特征在于，所述的试剂盒包含试验试管、移液管和微检测盘。
16.根据权利要求14所述的试剂盒，其特征在于，所述的试剂盒还包含颜色图谱。
CNB021589313A 2002-11-26 2002-12-27 测定昆虫对杀虫剂抗性的方法和试剂盒 Expired - Fee Related CN100498302C (zh)

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CN100442048C (zh) * 2005-03-28 2008-12-10 湖南文理学院 农药对捕食性天敌体内消化酶活性的快速检测方法

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MY141873A (en) * 2002-11-26 2010-07-16 Inst Medical Res Method for detecting elevated activity of oxidases in insects
KR102240047B1 (ko) * 2018-10-29 2021-04-15 경북대학교 산학협력단 포스핀 저항성 해충 진단용 바이오마커

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CA2114160A1 (en) * 1993-01-26 1994-07-27 Stephen C. Trowell Agents and method for identifying insects
US6517856B1 (en) * 1998-07-08 2003-02-11 Agdia Incorporated Insecticide resistance assay
US7081350B2 (en) * 2001-09-07 2006-07-25 Regents Of The University Of Minnesota Methods for identifying ecdysteroid synthesis inhibitors using the drosophila P450 enzyme shade
MY141873A (en) * 2002-11-26 2010-07-16 Inst Medical Res Method for detecting elevated activity of oxidases in insects

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100442048C (zh) * 2005-03-28 2008-12-10 湖南文理学院 农药对捕食性天敌体内消化酶活性的快速检测方法

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