美国进境樱桃果实中梨火疫病菌的检测

为了筛选快速、灵敏的梨火疫病菌检测方法,利用常规PCR、套式PCR和实时荧光PCR方法分别对美国进境的326批樱桃果实中梨火疫病菌进行检测。结果显示,3种PCR方法的检出率不同,不同引物或探针的检出率也存在差异。在常规PCR中,引物Ams3/Ams4c、P29A/P29B和PEANT1/PEANT2的检出率分别为35.28%、24.85%和16.87%;单管套式PCR和套式PCR的检出率分别为23.01%和50.61%;4种实时荧光PCR的检出率分别为17.48%(探针PA)、32.21%(探针Ams)、29.14%(探针ITS)和23.93%(SYBR GreenⅠ)。在所有试验方法中由引物P29A/P29B和PEANT1/PEANT2组成的套式PCR的检出率最高。检测结果证实了进境樱桃果实中存在梨火疫病菌DNA,套式PCR和常规PCR(引物Ams3/4c)可用于进境樱桃样品中梨火疫病菌的常规检测。     

进境玉米中玉米内州萎蔫病菌的PCR检测

 为了快速准确检测进境玉米样品中的玉米内州萎蔫病菌Clavibacter michiganensis subsp. nebraskensis(Cmn), 根据GenBank中Cmn的16S-23S序列设计引物CM1/CM4和引物PSM1/CM3。引物PSM1/CM3仅能从供试的4株Cmn菌株中扩增获得208 bp的预期产物, 而其他36株对照菌株均不能扩增出预期条带。灵敏度测试结果表明引物CM1/CM4和PSM1/CM3组合的巢式PCR方法的检测灵敏度高于常规PCR, 检测灵敏度可达40 fg DNA或6.8 CFU目标细菌。常规PCR和巢式PCR方法对进境美国玉米样品的阳性检出率分别为8%和24%, 试验结果表明所建立的PCR方法可用于玉米样品中Cmn的快速检测。     

向日葵黑茎病菌TaqMan-MGB探针实时荧光快速检测方法

 向日葵黑茎病菌是我国进境检疫性有害生物名录中的一种检疫性真菌。根据向日葵黑茎病菌及其近似种的ITS序列差异,设计并合成特异性引物和探针,建立了向日葵黑茎病菌的实时荧光PCR检测方法。特异性试验结果表明,该检测方法能特异性检测向日葵黑茎病菌;灵敏度试验结果表明,最低检测限量为20 μL反应体系中总DNA含量0.1 pg;实时荧光PCR优化反应条件为引物终浓度0.6 μmol·L^-1,探针终浓度0.3 μmol·L^-1。实际样品检测结果表明,该方法可用于疑似携带向日葵黑茎病菌样品的检测与初筛。此方法快速、灵敏,整个反应过程约1 h,检测过程完全闭管,无需PCR后续处理,为早期快速检测向日葵黑茎病菌提供了重要参考。     

进境苹果果实中梨火疫病菌的套式PCR检测

 针对进境商用苹果果实携带梨火疫病菌Erwinia amylovora数量有限的特点,选取源于病菌pEA29质粒的2对引物P29A/P29B和PEANT1/PEANT2配对组合成套式PCR,其检测灵敏度可达0.15 pg菌体DNA,检测灵敏度高于EPPO推荐的单管套式PCR方法和常规PCR方法。分别利用这3种PCR检测方法对美国、新西兰、日本和智利等国进境的166批苹果样品进行检测,3种检测方法的样品阳性率分别为53.6%、38.0%和8.4%,试验结果表明此套式PCR检测方法可用于进境商用苹果的梨火疫病菌快速检测。进境样品的检测结果证实了进境商用苹果果实中存在梨火疫病菌的可能性。     

基于TaqMan MGB探针的可可花瘿病菌快速检测方法

可可花瘿病菌是一种我国进境植物检疫性真菌?本文根据可可花瘿病菌EF1α基因的保守序列, 设计并合成1对特异性的实时荧光PCR引物和1条TaqMan MGB探针, 建立了可可花瘿病菌的实时荧光PCR检测方法?特异性试验结果表明, 该检测方法能够特异性检出可可花瘿病菌; 实时荧光PCR优化反应条件为引物终浓度0.2 μmol/L, 探针终浓度0.6 μmol/L; 灵敏度试验结果表明, 20 μL反应体系中可可花瘿病菌DNA含量最低检测限为10 pg; 重复性试验结果表明, 该检测方法的重复性和稳定性良好; 接种试验样品检测结果表明, 该方法可用于疑似携带可可花瘿病菌样品的检测与初筛?本文建立的方法具有良好的灵敏性?特异性和应用性, 为可可花瘿病菌早期快速检测提供了一种有效手段?     

基于LAMP的进境苜蓿种质鉴定技术的研究

本研究利用环介导等温扩增技术,建立了进境苜蓿种质的LAMP快速鉴定方法。选取叶绿体基因组中的条码基因mat K设计的1组特异性引物,进行反应条件优化,从而建立起了苜蓿种质的LAMP鉴定方法。该方法检测速度快,灵敏度比PCR法高10倍,可有效区分几种同为豆科的牧草;通过观察仪器输出的实时荧光曲线即可判断是否发生了扩增,大大提高了检测速度。本研究为进境苜蓿种质鉴定提供了一种较为快速、可行的方法。     

PCR方法检测美人蕉黄斑驳病毒

美人蕉黄斑驳病毒病是危害美人蕉的重要病害之一,我国目前尚无该病发生的报道.本文以健康和感病植株为材料,进行总DNA的提取和PCR扩增,从感病植株中扩增出与目的片段大小相符的产物,而健康植株中则未扩增出.PCR扩增产物经克隆、测序后证实,与已报道的美人蕉黄斑驳病毒(Cannayellow mottle virus,CaYMV)序列同源性高达97%～99%.经优化反应条件,建立了稳定的美人蕉黄斑驳病毒PCR检测方法.     

进境玉米中玉米内州萎蔫病菌PCR检测方法评估

为了准确检测玉米样品中玉米内州萎蔫病菌（Clavibacter michiganensis subsp. nebraskensis,Cmn）,减少测试过程中的假阳性和假阴性结果,利用15对Cmn特异性引物/探针分别测试了Cmn及其他菌株63株,并测试了食用、种用和饲用进境玉米样品300个。测试结果表明,常规PCR和实时荧光PCR方法阳性检出率最高的引物/探针分别是引物PSA-7/PSA-R和探针Cmn-MGB;巢式PCR不适用于样品中Cmn的检测;检测过程中引物Cmn-4F/Cmn-4R和探针rpsJ27P出现假阴性,引物CmnFP/CmnRP出现假阳性。根据测试结果,引物PSA-7/PSA-R和探针Cmn11-P可用于口岸进境玉米样品Cmn检测。     

利用常规PCR和实时荧光定量PCR检测杨梅凋萎病菌

凋萎病是近年来危害杨梅的主要病害。为了快速灵敏的检测杨梅凋萎病菌(Pestalotiopsis versicolor和P.microspora),本研究开发了常规PCR和SYBR Green实时荧光定量PCR技术各一套。利用P.versicolor(JN861773)和P.microspora(JN861776)的ITS1-5.8S rDNA-ITS2序列的相同部分设计引物对(Pvm1L/Pvm1R)。该引物对利用常规PCR技术能特异性扩增出杨梅凋萎病菌188 bp的目标产物而对照菌株则呈阴性。该常规PCR体系能够检测人工接种后21 d和田间自然发病的有病症杨梅组织中的凋萎病菌,检测下限是0.6×10~5拷贝数。利用Pvm1L/Pvm1R进行SYBR Green实时荧光定量PCR,检测灵敏度是常规PCR的100倍,检测下限是0.6×10~3拷贝数,能够检测出人工接种及田间已经感染但尚未表现症状的杨梅组织中的凋萎病菌。这两项技术简单、快速、灵敏特异性强,可以应用于杨梅凋萎病的诊断和苗木检疫。     

木尔坦棉花曲叶病毒RPA快速检测方法的建立

木尔坦棉花曲叶病毒Cotton leaf curl Multan virus(CLCuMuV)引起的病害是世界棉花生产上的毁灭性灾害,也是我国进境植物检疫性有害生物之一。因此,建立快速检测技术对CLCuMuV的检疫和防控具有重要意义。本研究根据CLCuMuV外壳蛋白(CP)基因序列设计引物,建立了该病毒的重组酶聚合酶等温扩增(recombinase polymerase amplification, RPA)检测方法,并评价了该方法的灵敏度和特异性,进一步测定了对田间疑似病样的检测准确性。结果表明,建立的RPA检测方法仅能从感染CLCuMuV的样品中扩增出目的条带,而感染同属的其他5种病毒的样品中未扩增出目的条带。该方法检测灵敏度是常规PCR的10倍,且对田间疑似病样的检出结果与PCR试验结果一致。因此,本研究所建立的CLCuMuV RPA快速检测方法具有特异、灵敏、准确、操作简便、无需特殊设备等优点,这些为CLCuMuV的快速检测提供了一种新技术。     

烟粉虱中甘薯双生病毒(sweepoviruses)半巢式PCR快速检测技术的建立与应用

甘薯双生病毒(sweepoviruses)是侵染甘薯的一类重要病毒,通过烟粉虱以持久方式传播,我国甘薯上至少存在8种甘薯双生病毒.本研究根据我国已报道的8种甘薯双生病毒基因组保守区设计了一组引物,建立了单头烟粉虱中甘薯双生病毒的半巢式PCR快速检测方法.特异性和灵敏性分析结果表明,半巢式PCR具有较高的特异性和灵敏性,...     

基于Ypt1基因为靶标的木香疫病病组织及病田土壤分子检测技术

A species-specific PCR assay was established for rapid and accurate detection of the oomycete pathogen Phytophthora tentaculata in diseased plant tissues and infected soil.A pair of species-specific primers Pt1/Pt2 were designed on the basis of Ras-related protein(Ypt1) gene sequences of the Phytophthora species.PCR amplification with the Pt primers resulted in a 386 bp product only from isolates of P.tentaculata.The detection threshold with Pt primers was 100 pg of genomic DNA.A nested PCR procedure was developed using Ypt1F/Ypt1R as the first-round amplification primers and Pt1/Pt2 as the second-round primers,which increased the detection sensitivity 100-fold to 1 pg.PCR using these Pt primers can also be used to detect P.tentaculata in naturally infected plant tissues and soil.The PCR-based method developed in this study provides a rapid and sensitive tool for detection of P.tentaculata.     

Detection of Xanthomonas citri subsp. citri from field samples using single‐tube nested PCR

A single‐tube nested PCR was developed for detection of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. The assay targets the pthA gene of Xcc and utilizes different annealing temperatures for the two primer pairs. It reliably detected as few as 1·0 × 10² Xcc cells, and was unaffected by the presence of PCR inhibitors. It was 10‐fold and 8500‐fold more sensitive than standard PCR and ELISA, respectively. Increased sensitivity was also achieved via the use of a washing method for DNA extraction, as opposed to direct extraction from leaf tissue. When evaluated for Xcc detection in 90 samples collected from affected pomelo orchards, the single‐tube nested PCR was superior to standard PCR, detecting the pathogen in 67 vs. 54 samples. It was also able to detect Xcc from samples with and without symptoms. This assay can be used as a rapid and sensitive technique for routine Xcc detection in field samples for surveillance of citrus canker.     

Reliable RT-PCR detection of Apple stem pitting virus in pome fruits and its association with quince fruit deformation disease

In this study a spot nested RT-PCR assay was developed for the detection of Apple stem pitting virus (ASPV). A one step RT-PCR for the generic detection of foveaviruses using degenerate primers that target a conserved region of the RNA-dependent RNA polymerase (RdRp) gene was followed by a nested PCR that amplifies a 312 bp ASPV specific product. The method is rapid, simple and displays high sensitivity and broad detection range, overcoming the virus molecular variability. The optimum sampling conditions for reliable virus detection were also investigated. ASPV was detected throughout the year in different plant tissues of affected trees, thus the method could be used for routine screening and in certification schemes of pome fruits. ASPV was detected in quince orchards in Greece in all trees that were tested, showing a fruit deformation disorder. Sequencing and phylogenetic analysis of amplicons generated by RT-PCR from plant tissue affected with the deformation disease indicated that the agent responsible was a variant of ASPV.     

Development and evaluation of specific PCR and LAMP assays for the rapid detection of Phytophthora melonis

Phytophthora melonis is a widespread and devastating pathogen for the Cucurbitaceae family. Early and accurate detection of P. melonis is essential to control the disease in the field. To establish a simple, visual, and rapid detection system for P. melonis, we developed nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) systems based on the Ras-related protein (Ypt1) gene. All 36 isolates of P. melonis, from geographically distinct counties in China, yielded positive detection results on LAMP or nested PCR assays. No cross reaction was observed with other oomycetes or fungal pathogens. A sensitivity assay showed that both methods had a detection limit of 10 fg genomic DNA. We also detected P. melonis in diseased cucumber tissues and soils, and evaluated positive detection rates using LAMP, nested PCR, and conventional isolation methods. The results suggest that the LAMP assay has the greatest potential for active detection of P. melonis in regions that are at risk of contracting the disease, and for use in resource-poor settings.     

三种PCR方法检测柑橘黄龙病菌的效果比较

为了比较常规PCR、巢式PCR和实时荧光定量PCR方法在大田检测中对柑橘黄龙病(Huanglongbing, HLB)的检测效果, 首先比较了3种检测方法对柑橘黄龙病菌检测的灵敏度, 结果发现：3种检测方法的灵敏度依次为常规PCR<巢式PCR<实时荧光定量PCR。运用3种检测方法对广东5个柑橘品种上的189个黄龙病疑似病样进行检测, 结果发现：黄龙病检出率依次为常规PCR<巢式PCR<实时荧光定量PCR。研究表明：常规PCR适合以较低成本大规模检测黄龙病; 实时荧光定量PCR具有最大的检测灵敏度; 巢式PCR检测技术同时具有前两者的一些优点, 但操作较复杂, 适合技术熟练的研究者使用。     

雪松疫霉(Phytophthora lateralis)的快速分子检测

由雪松疫霉(Phytophthora lateralis)引起的疫病是一类植物检疫性病害。为建立该病原菌的快速检测技术,本文比较分析了雪松疫霉和其他疫霉的tRNA序列,在此基础上设计了一对检测雪松疫霉的特异性引物T1/T2,该对引物从雪松疫霉中扩增得到1条192 bp的条带,而其他15种疫霉和其他真菌菌株均无扩增条带,表明该对引物对雪松疫霉具有特异性。在25μL PCR反应体系中,引物T1/T2检测灵敏度为10 pg基因组DNA;而以引物T3/T4和T1/T2进行巢式PCR扩增,能够检测到1 fg基因组DNA,使检测灵敏度提高了10 000倍。该检测体系对灭菌水中游动孢子的检测灵敏度可达0.5个游动孢子,对人工接种发病的植物组织能够特异性地检测到该病原菌。此外,进一步建立了该病原菌的实时荧光定量PCR检测体系。     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

犬细小病毒病PCR检测方法的建立及应用

犬细小病毒病感染是由犬细小病毒（cPv）引起犬只死亡的最重要传染病之一,目前诊断该病的方法主要是胶体金快速检测试板法,虽然检测方法较简便快速,但其敏感性较差。通过对NCBI网站GenBank发表的CPV-2基因组序列的分析,选择该病毒VP2基因保守序列设计引物,通过对阳性病料扩增及扩增产物测序,与NCBI发表的相关基因对比,同源性在99．8％-100％,成功建立了特异性、灵敏度高的PCR方法,最低只需2．5PgDNA模板。在对28例可疑CPV病例检测中,本方法检出25例阳性、3例阴性,阳性率为89．28％;胶体金检测板检测出20例阳性、8例阴性,阳性率为71．42％,证实PCR方法比胶体金检测更敏感。