转基因抗虫耐除草剂玉米MON87411精准定量检测方法的建立

【目的】抗虫耐除草剂玉米MON87411是孟山都远东有限公司利用农杆菌介导方法研发的玉米转化体,已获得我国进口用作加工原料的农业转基因生物安全证书。为满足生物安全监管的要求,亟需建立该转化体的定量检测方法。【方法】根据抗虫耐除草剂玉米MON87411的两端旁侧序列信息设计引物和Taqman探针,进行引物筛选、特异性检测、PCR体系优化、标准曲线建立、正确度及精密度检测、检出限及定量限测试、微滴数字PCR方法验证等。【结果】该方法能特异、定量地检测出抗虫耐除草剂玉米MON87411转化体成分,检出限低至10拷贝,定量限为40拷贝。对测试样品定值准确,经微滴数字PCR方法验证结果一致。【结论】本方法为该新品种转基因玉米品系的精准定量提供了一种新的检测方法,为生物安全监管提供了有效的技术支撑。     

双重数字PCR在转基因大豆检测中的应用

利用微滴式数字PCR(droplet digital PCR, ddPCR)平台建立针对MON87705、MON87769、DP356043三种转基因大豆中外源基因的双重PCR检测方法。利用双重数字PCR方法检测特异性、定量范围等参数,优化所用引物探针组合及实验体系程序,检测外源基因与内标准基因的拷贝数。结果表明,所用引物探针组合在数字PCR方法中仅对目标大豆品系有荧光信号,具有特异性,可用于转基因大豆品系的筛选与鉴别。检测了大豆的转基因成分含量,结果与材料标准品参数基本一致,并根据结果设定定量检测限为0.5%,定性检测限为0.05%,可满足低纯度样品检测的需求。双重数字PCR体系能够准确且稳定的满足实际检测需要,在实际应用上具有良好的发展前景。     

转基因玉米NK603品系特异定量PCR检测方法的建立

目的:建立转基因玉米NK603品系特异定量PCR检测方法,为转基因玉米NK603提供科学的定量检测依据。方法:根据转基因玉米NK603外源基因的旁侧序列设计引物和Taqman探针,建立转基因玉米NK603品系特异定量PCR检测方法,并采用该法检测2%含量的NK603标准品(不确定度为10%)。结果:采用构建的方法获得标准曲线斜率为-3.6~-3.1,相关系数大于0.99,扩增效率为100.2%,在90%~110%范围内;样品检测结果(1.9%)接近已知含量(2%,不确定度为10%)。结论:建立的转基因玉米NK603品系特异定量PCR检测方法的准确度较高,可在日常检验中推广应用。     

转基因大豆GTS40-3-2转化事件特异性PCR检测

GTS40-3-2是抗草甘膦转基因大豆,为建立GTS40-3-2大豆转化体特异性PCR检测方法,本研究以GTS40-3-2标准品为实验材料,根据已公布转基因大豆GTS40-3-2基因与大豆基因组连接序列信息,利用Primer5.0软件设计了5对品系特异性引物,对每对引物进行了退火温度、特异性及扩增效率的PCR检测,结果显示,5对特异性引物均能够从GTS40-3-2中扩增出大小约279bp、238bp、470bp、490bp和257bp的预期产物,可用于特异性检测转基因大豆GTS40-3-2转化事件。以转基因大豆GTS40-3-2含量为5%、2%、1%、0.5%和0.1%的标准品进行PCR灵敏度检测,结果表明5对引物的检测灵敏度均能达到0.1%。通过荧光定量PCR对5对特异性引物的Ct值与溶解曲线比较,最后选择出RRS2引物对为转基因大豆GTS40-3-2品系特异性检测的最适引物。本文结果将为我国未来转基因生物产品成分检测提供科学合理的实验参考。     

转Cry1Ab基因水稻分子特征及其特异性PCR检测方法

转Cry1Ab基因水稻Bt01为一种新型的转基因水稻,文章首先利用Southern blotting验证了外源基因Cry1Ab转入了Bt01中,且为单拷贝,再利用TAIL-PCR方法获得了其插入位点信息,根据获得的Bt01的5′端插入位点序列,设计了相应的定性与定量PCR检测体系的引物及探针,实验结果显示,定性PCR检测体系的最低检测极限(LOD)为10个拷贝,定量PCR检测体系的LOD为5拷贝,最低定量极限(LOQ)为10拷贝。同时为了验证建立的定量PCR体系的准确性,利用该体系检测已知转基因水稻Bt01含量分别为3%和0.5%的样品,定量结果分别为2.7%和0.47%。研究结果表明,该转化体特异性定性与定量检测方法具有高度的特异性和良好的灵敏性,为转基因水稻Bt01的身份识别和检测提供了有效的方法。     

转Cry1Ab基因水稻分子特征及其特异性PCR检测方法

转Cry1Ab基因水稻Bt01为一种新型的转基因水稻, 文章首先利用Southern blotting验证了外源基因Cry1Ab转入了Bt01中, 且为单拷贝, 再利用TAIL-PCR方法获得了其插入位点信息, 根据获得的Bt01的5′端插入位点序列, 设计了相应的定性与定量PCR检测体系的引物及探针, 实验结果显示, 定性PCR检测体系的最低检测极限(LOD)为10个拷贝, 定量PCR检测体系的LOD为5拷贝, 最低定量极限(LOQ)为10拷贝。同时为了验证建立的定量PCR体系的准确性, 利用该体系检测已知转基因水稻Bt01含量分别为3%和0.5%的样品, 定量结果分别为2.7%和0.47%。研究结果表明, 该转化体特异性定性与定量检测方法具有高度的特异性和良好的灵敏性, 为转基因水稻Bt01的身份识别和检测提供了有效的方法。     

转基因玉米双抗12-5转化体特异性PCR方法验证结果分析

抗虫和耐除草剂玉米双抗12-5是我国自主研发的转基因品种,该品种于2020年1月21日获得农业转基因生物安全证书,具有广阔的应用前景。转化体特异性PCR方法是进行转基因生物安全监管的最有效的技术手段之一,可以对转基因产品进行身份鉴定。本研究组织8家实验室对研发单位提供的的双抗12-5转化体特异性定性、定量PCR方法进行了验证,验证结果显示定性与定量PCR检测方法均具有稳定性好、特异性强和灵敏度高的特点,定量PCR方法能精确地定量检测质量分数为5%和0.5%的双抗12-5样品,并且具有良好的重复性和再现性,符合相关标准的要求。本研究有助于后续标准方法的建立和完善,为我国转基因生物安全监管提供技术支撑和决策依据。     

转基因玉米双抗12-5转化体特异性PCR方法验证结果分析

抗虫和耐除草剂玉米双抗12-5是我国自主研发的转基因品种,该品种于2020年1月21日获得农业转基因生物安全证书,具有广阔的应用前景。转化体特异性PCR方法是进行转基因生物安全监管的最有效的技术手段之一,可以对转基因产品进行身份鉴定。本研究组织8家实验室对研发单位提供的的双抗12-5转化体特异性定性、定量PCR方法进行了验证,验证结果显示定性与定量PCR检测方法均具有稳定性好、特异性强和灵敏度高的特点,定量PCR方法能精确地定量检测质量分数为5%和0.5%的双抗12-5样品,并且具有良好的重复性和再现性,符合相关标准的要求。本研究有助于后续标准方法的建立和完善,为我国转基因生物安全监管提供技术支撑和决策依据。     

转基因水稻“科丰6号”实时荧光PCR定性定量检测方法研究

旨在建立转基因水稻"科丰6号"外源基因和边界序列的实时荧光PCR检测方法,为科丰6号定性定量检测提供技术支持。根据外源基因和边界序列信息,设计实时荧光PCR探针引物,优化体系,对不同转基因产品和不同转基因含量的"科丰6号"水稻进行检测。结果显示,所设计的引物探针具有很好的特异性,与其他转基因水稻品系、转基因玉米、转基因棉花、转基因番茄和非转基因水稻均无非特异性反应,对转基因水稻"科丰6号"的检测灵敏度达到0.01%。建立的科丰6号实时荧光PCR检测方法重复性好、灵敏度高,能够达到目前国际上转基因产品定量检测的标准,为该水稻品系的定性定量检测提供技术支持。     

耐除草剂玉米G1105E-823C定性检测方法

转基因耐除草剂玉米G1105E-823C是经过改造的转mG2-aroA基因耐草甘膦玉米新品系,具有更高的草甘膦耐受性,目前已完成生产性试验,具有重要的产业化应用前景。但目前尚无针对G1105E-823C的转化体特异性检测方法的相关报道,这十分不利于对该品系的检测及监管。基于此,以G1105E-823C转化体特异性序列为靶标,建立了转基因耐除草剂玉米G1105E-823C的普通PCR和实时荧光PCR定性检测方法。结果表明,2种方法均能检测出转基因耐除草剂玉米G1105E-823C转化体成分,且具有较高的特异性。普通PCR检测方法检出限达0.1%,实时荧光PCR检测方法检出限达0.05%。研究建立的2种定性检测方法为转基因耐除草剂玉米G1105E-823C的精准检测提供了新的技术手段,可为农业转基因监管提供技术支撑。     

抗虫玉米CM8101定性检测方法的建立

转基因抗虫玉米CM8101是经人工改造合成的转Bt抗虫基因Cry1Ab?Ma转基因玉米新品系,由我国自主研发,具有更优良的抗虫性,目前已进入生产性试验阶段,具有广阔的产业化应用前景。依据CM8101的5′端旁侧序列信息,设计并筛选出最佳引物探针组合,通过普通PCR和实时荧光PCR技术,建立了转基因抗虫玉米CM8101的两种特异性定性检测方法。结果显示,普通PCR检测方法检出限达0.1%,实时荧光PCR检测方法检出限达0.05%。这两种定性检测方法的建立为今后准确高效检测转基因抗虫玉米CM8101及其产品提供了新的参考方法。     

Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.     

Quantitative real-time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.     

肠出血性大肠杆菌(O157：H7)的特异性荧光探针检测方法的建立

目的:建立一种real-time PCR,快速准确检测肠出血性大肠杆菌O157:H7。方法:以肠出血性大肠杆菌0157:H7 rfbE为待检靶基因,设计一对引物和一条Taqman探针,探针5’端用FAM基团标记,3’端用TAMRA标记。通过重组质粒的构建,建立并优化了大肠杆菌0157:H7的荧光定量PCR检测方法。结果:在人工污染样本无需富集的情况下,检测的最低DNA浓度是10拷贝/反应(3CFU/mL);特异性检测实验中,0157菌株检测结果均为rfbE阳性,而非0157:H7菌株检测结果均为阴性;重复性实验中,批内、批间变异系数均小于3%。结论:实验结果显示此荧光定量PCR方法特异性、灵敏度高,重复性好,可对分离的可疑大肠杆菌0157:H7菌株进行快速鉴定。     

O1群霍乱弧菌实时荧光定量PCR快速检测方法的建立

目的:建立针对O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,并进行模拟粪便标本的检测评价。方法:根据O1群霍乱弧菌O抗原编码基因rfb的特异性序列设计引物和TaqMan探针,建立检测O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,对所建立的方法分别进行实验室内的灵敏度及特异性评价;将O1群霍乱弧菌灭活菌株悬液倍比稀释后与健康成人新鲜粪便混匀,制备成模拟带菌者粪便标本,提取DNA,进行Taq-Man PCR检测,用以评价该方法。结果:建立了快速检测O1群霍乱弧菌的实时荧光定量TaqMan PCR方法,灵敏度为每反应体系104拷贝;该方法对其他14种肠道菌DNA没有扩增;该方法对模拟粪便标本的检测灵敏度为每反应体系102 CFU。结论:建立了一种快速、高效检测O1群霍乱弧菌的荧光定量PCR检测方法,该方法可用于O1群霍乱弧菌临床粪便标本的检测。     

Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize

This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.     

Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5' nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to >/=10(2) cells ml(-1) was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.     

GA21转基因玉米实时荧光PCR检测方法的建立

成功建立了实时荧光PCR鉴定检测转基因玉米GA21品系的方法。该方法通过GA21玉米品系的OTPmEPSPS边界的270bp和133bp靶序列,设计品系特异性检测引物和探针,同时针对Pactin1mEPSPS边界的430bp靶序列设计品系特异性检测引物,应用实时荧光PCR和PCR技术,特异性检测GA21玉米品系。结果表明,应用实时荧光PCR的TaqMan探针技术检测转基因作物边界序列,不仅可以达到品系鉴定的目的,而且该方法和常规PCR比特异性强,简便快速,同时实验采用完全闭管检测,又降低了污染机会,为转基因作物的品系鉴定检测提供了新方法。