利用DNA分子指纹图谱技术检测小麦粉中害虫的研究
|
摘要
为开发快速检测我国成品粮中害虫的技术,研究了DNA分子指纹图谱技术检测小麦粉中主要害虫的碎片和虫卵的含量。结果表明,通过对害虫的延长因子EF1基因片段引物的筛选和PCR条件的选择,确定了有效地检测出来小麦粉中的赤拟谷盗、杂拟谷盗和锯谷盗的成虫碎片、幼虫和卵通用引物和PCR扩增条件。通过改良的PCR技术可在4 h内快速检测杂拟谷盗、锯谷盗的卵、幼虫和成虫碎片。最低检出限为可检测出小麦粉中0. 05%(m/m)卵和幼虫、1%成虫的碎片,灵敏度超过了国际标准。DNA指纹图谱中电泳条带亮度(Y)与被检测的不同害虫碎片含量(X)成正相关,回归方程为Y=858. 36X+74. 540相关系数R为0.961 9。利用该技术检测随机取样的小麦粉中成虫的碎片,检测的准确率最高可达80. 56%。结果说明了利用DNA分子指纹图谱技术可快速检测出小麦粉中害虫的碎片、幼虫和卵含量,并可预测小麦粉中害虫的感染程度。本研究为开发快速小麦粉中害虫的快速检测技术具有重要的参考意义。
To develop the technology of fast detection of insects in our processed grain, the molecular fingerprint technique was used to detect quickly main fragments and eggs of insect in wheat flour.The results showed that the universal primers and condition of PCR were confirmed that could be used to detect the adult fragments, larvae and eggs of Tribolium castaneum, T.confusum and Oryzaephilus surinamensis by selecting the primers for insect elongation factor gene EF1 and PCR condition.Modified PCR technology could quickly detect the eggs 、larvae and adult fragments of T.confusum and 0.surinamensis in wheat flour in 4 hours.The lowest detection limitation for eggs and larvae of wheat flour is 0.05%, for adults fragments is 1 %.Sensitivity of detection exceeds the international standard.There was a positive correlation between brightness of DNA agarose gel electrophoresis banding( Y) and adult fragments contents in flour(X),and the regression equation is Y = 858.36 X + 74.540(R=0.961 9).The highest prediction accuracy with DNA fingerprint technique to detect adult fragments within the random sampling of flour was80.56%.Results showed that the DNA molecular fingerprinting technology could be used to rapidly detect contents of insect fragments, larval and eggs in wheat flour and predict the infection degree of insect in wheat flour.Our research had significant value to explore the rapid detection technique for insect in wheat flour.
To develop the technology of fast detection of insects in our processed grain, the molecular fingerprint technique was used to detect quickly main fragments and eggs of insect in wheat flour.The results showed that the universal primers and condition of PCR were confirmed that could be used to detect the adult fragments, larvae and eggs of Tribolium castaneum, T.confusum and Oryzaephilus surinamensis by selecting the primers for insect elongation factor gene EF1 and PCR condition.Modified PCR technology could quickly detect the eggs 、larvae and adult fragments of T.confusum and 0.surinamensis in wheat flour in 4 hours.The lowest detection limitation for eggs and larvae of wheat flour is 0.05%, for adults fragments is 1 %.Sensitivity of detection exceeds the international standard.There was a positive correlation between brightness of DNA agarose gel electrophoresis banding( Y) and adult fragments contents in flour(X),and the regression equation is Y = 858.36 X + 74.540(R=0.961 9).The highest prediction accuracy with DNA fingerprint technique to detect adult fragments within the random sampling of flour was80.56%.Results showed that the DNA molecular fingerprinting technology could be used to rapidly detect contents of insect fragments, larval and eggs in wheat flour and predict the infection degree of insect in wheat flour.Our research had significant value to explore the rapid detection technique for insect in wheat flour.
引文
[1] Health Canada, Health Protection BranchStandards and Guidelines for the General Cleanliness of Food-an Overview[J].Canadian Institute of Food Science and Technology Journal. 1999
[2] GLAZE L E, BRYCE J R. Extraction of light filth from whole wheat flour. Floatation method:collaborative study[J].Journal of AOAC International, 1994,77:1150-1152
[3]刘涛,石宇.夏季面粉虫害防控的一些措施[J].现代面粉工业,2015,2:11LIU Tao,SHI Yu. Some measure to control insect pest in flour in summer[J]. Modern Flour Milling Industry, 2015,2:11
[4]张景渠.环境条件对小麦及小麦粉质量的影响[J].粮食加工2017, 42(1):16-18ZHANG Jingqu. Effect of environment condition on wheat and flour quality[J]. Grain Processing, 2017, 42(1):16-18
[5]AACC, Official methods of Analysis[S]. 9th Edit. American Association of Cereal Chemists. Minnesota. USA. 1955
[6] SMITH M PAYNE R B. Re-examination of effect of paracetamol on serum uric acid measured by phosphotungstic acid reduction[J]. Annals of Clinical Biochemistry, 1979, 16(2):96-99
[7]LEELAJA B C, RAJASHEKAR Y, Rajendran S. Detection of eggs of stored-product insects in flour with staining techniques[J]. Journal of Stored Products Research, 2007, 43:206-210
[8] MOTONAKA J, MIYATA K, Fauliner L R. Micro EnzymeSensor with Osimium Complex and a Porous Carbon for Measuring Uric Acid[J]. Analytical Letters, 1994,27-31
[9]刘凤杰,何向楠,王争艳,等.小麦粉中虫卵的快速检测技术的研究与比较[J].中国粮油学报,2014, 29(12):119-123LIU Fengjie, HE Xiangnan, WANG Zhengyan et al. Comparison of the rapid methods for detection the insect ovum in the flour[J]. Journal of the Chinese Cereals and Oils Association, 2014, 29(12):119-123
[10]袁园,刘凤杰,王争艳,等.高效液相色谱法检测小麦粉感染害虫后尿酸含量的研究[J].河南工业大学学报,2015, 36(4):20-26YUAN Yuan, LIU Fengjie, WANG Zhengyan, et al. Study on detection of uric acid content in wheat flours infested by pest insects in high performance liquid chromatography(HPLC)[J]. Journal of Henan University of Technology(Natural Science Edition), 2015, 36(4):20-26
[11]李钟淑,金粉玉,王士勇,等.DNA指纹技术在动物亲子鉴定中的应用[J].延边大学农学学报,2003, 25(2):131-133,148LI Zhongshu, JIN Fenyu, WANG Shiyong, et al. Application of DNA fingerprint technique for animal parentage identification[J]. Journal of Agricultural Science Yanbian University, 2003, 25(2):131-133,148
[12] PEREZ-Mendoza J, THRONE J E, DOWELL F E, et al.Detection of insect fragments in wheat flour by near-infrared spectroscopy[J]. Journal of Stored Products Research,2003, 39:305-312
[13] BALASUBRANMANIAN A, JAY AS D S, FERNABDO W G,et al. Sensitivity analysis of DNA fingerprinting technique for detecting insect fragments in wheat flour[J]. Canadian Biosystems Engineering, 2007,49:41-45
[14] BENNETT J. DNA-based techniques for control of rice insects and diseases transformation, gene tagging, and DNA fingerprinting[J]. AGRIS, 2000:147-157.
[2] GLAZE L E, BRYCE J R. Extraction of light filth from whole wheat flour. Floatation method:collaborative study[J].Journal of AOAC International, 1994,77:1150-1152
[3]刘涛,石宇.夏季面粉虫害防控的一些措施[J].现代面粉工业,2015,2:11LIU Tao,SHI Yu. Some measure to control insect pest in flour in summer[J]. Modern Flour Milling Industry, 2015,2:11
[4]张景渠.环境条件对小麦及小麦粉质量的影响[J].粮食加工2017, 42(1):16-18ZHANG Jingqu. Effect of environment condition on wheat and flour quality[J]. Grain Processing, 2017, 42(1):16-18
[5]AACC, Official methods of Analysis[S]. 9th Edit. American Association of Cereal Chemists. Minnesota. USA. 1955
[6] SMITH M PAYNE R B. Re-examination of effect of paracetamol on serum uric acid measured by phosphotungstic acid reduction[J]. Annals of Clinical Biochemistry, 1979, 16(2):96-99
[7]LEELAJA B C, RAJASHEKAR Y, Rajendran S. Detection of eggs of stored-product insects in flour with staining techniques[J]. Journal of Stored Products Research, 2007, 43:206-210
[8] MOTONAKA J, MIYATA K, Fauliner L R. Micro EnzymeSensor with Osimium Complex and a Porous Carbon for Measuring Uric Acid[J]. Analytical Letters, 1994,27-31
[9]刘凤杰,何向楠,王争艳,等.小麦粉中虫卵的快速检测技术的研究与比较[J].中国粮油学报,2014, 29(12):119-123LIU Fengjie, HE Xiangnan, WANG Zhengyan et al. Comparison of the rapid methods for detection the insect ovum in the flour[J]. Journal of the Chinese Cereals and Oils Association, 2014, 29(12):119-123
[10]袁园,刘凤杰,王争艳,等.高效液相色谱法检测小麦粉感染害虫后尿酸含量的研究[J].河南工业大学学报,2015, 36(4):20-26YUAN Yuan, LIU Fengjie, WANG Zhengyan, et al. Study on detection of uric acid content in wheat flours infested by pest insects in high performance liquid chromatography(HPLC)[J]. Journal of Henan University of Technology(Natural Science Edition), 2015, 36(4):20-26
[11]李钟淑,金粉玉,王士勇,等.DNA指纹技术在动物亲子鉴定中的应用[J].延边大学农学学报,2003, 25(2):131-133,148LI Zhongshu, JIN Fenyu, WANG Shiyong, et al. Application of DNA fingerprint technique for animal parentage identification[J]. Journal of Agricultural Science Yanbian University, 2003, 25(2):131-133,148
[12] PEREZ-Mendoza J, THRONE J E, DOWELL F E, et al.Detection of insect fragments in wheat flour by near-infrared spectroscopy[J]. Journal of Stored Products Research,2003, 39:305-312
[13] BALASUBRANMANIAN A, JAY AS D S, FERNABDO W G,et al. Sensitivity analysis of DNA fingerprinting technique for detecting insect fragments in wheat flour[J]. Canadian Biosystems Engineering, 2007,49:41-45
[14] BENNETT J. DNA-based techniques for control of rice insects and diseases transformation, gene tagging, and DNA fingerprinting[J]. AGRIS, 2000:147-157.