公开(公告)号：US06316195B1

公开(公告)日：2001-11-13

申请号：US09471016

申请日：1999-12-23

申请人： Reid D. Frederick ,  Paul W. Tooley ,  Morris R. Bonde ,  David A. Knorr ,  Gary L. Peterson ,  Norman W. Schaad

发明人： Reid D. Frederick ,  Paul W. Tooley ,  Morris R. Bonde ,  David A. Knorr ,  Gary L. Peterson ,  Norman W. Schaad

IPC分类号： C07H2104

CPC分类号： C12Q1/6895

摘要： Karnal bunt of wheat is caused by Tilletia indica Mitra. Recently, teliospores morphologically resembling T. indica were isolated from bunted ryegrass seeds and wheat seed washes. Previously developed PCR assays failed to differentiate T. indica from the newly discovered ryegrass pathogen, T. walkeri. The nucleotide sequence of a 2.3-kb region of mitochondrial DNA, previously amplified by PCR only from T. indica, was determined for three isolates of T. indica and for three isolates of T. walkeri. There was greater than 99% identity within either the T. indica group or the T. walkeri group of isolates, whereas there was approximately 3% divergence between isolates of these two Tilletia species. Five sets of PCR primers were made specific to T. indica, and three sets were designed specifically for T. walkeri based upon nucleotide differences within the mtDNA region. In addition, a 212 bp amplicon was developed as a target sequence in a fluorogenic 5′ nuclease PCR assay using the TaqMan system for the detection and discrimination of T. indica and T. walkeri.

摘要翻译： 小麦的Karnal b is是由Tilletia indica Mitra造成的。 最近，从from ed黑麦草种子和小麦种子洗涤中分离出形态上类似于indic，的eli豆。 以前开发的PCR测定法不能将T. indica与新发现的黑麦草病原体T. walkeri区分开来。 线粒体DNA的2.3-kb区域的核苷酸序列，先前仅通过PCR扩增，来自T. indica，测定了三种indic three分离株和三个分离的T. walkeri的分离株。 indic a a i i i i i i i。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。 基于t a特异性的5组PCR引物，基于mtDNA区域内的核苷酸差异，专门设计了3组。 此外，在荧光5'核酸酶PCR测定中，使用TaqMan系统发现212bp扩增子作为靶序列，用于检测和鉴别T. indica和T. walkeri。

公开(公告)号：US5811240A

公开(公告)日：1998-09-22

申请号：US773739

申请日：1996-12-24

申请人： Marisa A. S. V. Ferreira ,  Paul W. Tooley ,  Efstathios Hatziloukas ,  Norman W. Schaad ,  Morris R. Bonde

发明人： Marisa A. S. V. Ferreira ,  Paul W. Tooley ,  Efstathios Hatziloukas ,  Norman W. Schaad ,  Morris R. Bonde

IPC分类号： C07K14/375 ,  C12Q1/68 ,  C07H21/04 ,  C12P19/34

CPC分类号： C12Q1/6895 ,  C07K14/375

摘要： Mitochondrial DNA of five isolates of Tilletia indica was isolated and digested with several restriction enzymes. A 2.3 kb- EcoRI fragment was chosen, cloned, and shown to hybridize with total DNA restricted with EcoRI from T. indica and not from a morphologically similar smut fungus, T. barclayana. The clone was partially sequenced, primers were designed and tested under high-stringency conditions in PCR assays. The primer pair Ti1/Ti4 amplified a 2.3 kb fragment from total DNA of 17 T. indica isolates from India, Pakistan and Mexico. DNA from 25 isolates of other smut fungi (T. barclayana, T. foetida, T. caries, T. fusca and T. controversa) did not produce any bands as detected by ethidium bromide-stained agarose gels and Southern hybridizations. Sensitivity of the assay was determined and increased by using a single nested primer in a second round of amplification, so that 1 pg of total mycelial DNA could be detected. The results indicated that the primers which originated from a cloned mtDNA sequence can be used to differentiate T. indica from other Tilletia species and have the potential to identify teliospores contaminating wheat seeds.

摘要翻译： 分离并分离出五种分离株Tilletia indica的线粒体DNA，并用几种限制酶消化。 选择2.3kb-EcoRI片段，克隆并显示与来自indic a的EcoRI限制的总DNA杂交，而不是来自形态学上类似的真菌真菌T.barclayana。 将克隆部分测序，在PCR测定中在高严格条件下设计和测试引物。 引物对Ti1 / Ti4从印度，巴基斯坦和墨西哥的17株indic a分离株的总DNA扩增了2.3kb片段。 来自其他真菌真菌（T. barclayana，T. foetida，T.caries，T. fusca和T. controversa）的25个分离株的DNA不产生由溴化乙锭染色的琼脂糖凝胶和Southern杂交检测到的任何条带。 通过在第二轮扩增中使用单个嵌套引物测定和增加测定的灵敏度，使得可以检测到1μg的总菌丝体DNA。 结果表明，源自克隆的mtDNA序列的引物可用于区分indic from与其他ia ia属物种，并有潜力鉴定eli豆孢子污染小麦种子。

公开(公告)号：US5776686A

公开(公告)日：1998-07-07

申请号：US772961

申请日：1996-12-24

申请人： Oney P. Smith ,  Gary L. Peterson ,  Raymond J. Beck ,  Morris R. Bonde ,  Norman W. Schaad

发明人： Oney P. Smith ,  Gary L. Peterson ,  Raymond J. Beck ,  Morris R. Bonde ,  Norman W. Schaad

IPC分类号： C12Q1/68 ,  C07H21/02 ,  C07H21/04 ,  C12N15/00

CPC分类号： C12Q1/6895

摘要： The polymerase chain reaction (PCR) was used to identify Tilletia indica, the causal agent of Karnal bunt of wheat. The method uses two sets of oligonucleotide primers developed by sequence analysis of cloned Dra I fragments of mitochondrial DNA of T. indica. The primer pair TI17M1 (5'-TCCCCTTGGATCAGAACGTA-3') and TI17M2 (5'-AGAAGTCTAACTCCCCCCTCT-3'), derived from clone pTI-MD17, amplified a single 825-bp product from all isolates of T. indica and no products for other Tilletia species. In addition, the primer pair TI57M1 (5'-TTTTCCCTCTCTC-CTTTTTTCA-3') and TI57M2 (5'-AGCAAAGACAAAGTAGGCTTCC-3'), derived from clone pTI-MD57, produced a product of 118 bp which was unique to T. indica.

摘要翻译： 使用聚合酶链反应（PCR）来鉴定小麦的Karnal bunt的致病剂Tilletia indica。 该方法采用两组寡核苷酸引物，通过对indic of线粒体DNA的克隆DraⅠ片段进行序列分析而开发。 衍生自克隆pTI-MD17的引物对TI17M1（5'-TCCCCTTGGATCAGAACGTA-3'）和TI17M2（5'-AGAAGTCTAACTCCCCCCTCT-3'）扩增了来自indic from的所有分离株的单个825bp产物， 其他Tilletia种。 另外，来自克隆pTI-MD57的引物对TI57M1（5'-TTTTCCCTCTCTC-CTTTTTTCA-3'）和TI57M2（5'-AGCAAAGACAAAGTAGGCTTCC-3'）产生118bp的产物，其是T. indica 。

公开(公告)号：US6146834A

公开(公告)日：2000-11-14

申请号：US393877

申请日：1999-09-10

申请人： Norman W. Schaad ,  Wan-Yeob Song ,  Efstathios Hatziloukas

发明人： Norman W. Schaad ,  Wan-Yeob Song ,  Efstathios Hatziloukas

IPC分类号： C12Q1/68

CPC分类号： C12Q1/689 ,  C12Q2561/101

摘要： We sequenced a 625 and 617 bp fragment of the inner spacer region of 16S-23S rDNA of a strain of Acidovorax avenae representing pathogens from several hosts, including foxtail, oats, corn, rice, millet, sugarcane, orchid, and watermelon and a strain of A. avenae subsp. citrulli pathogenic only to watermelon, respectively, for the purpose of designing PCR primers for their identification. These plant pathogens were previously considered as non-fluorescent pseudomonads and have been recently reclassified as Acidovorax avenae subsp. avenae, A. avenae subsp. cattleyae, and A. avenae subsp. citrulli. Several sets of primers were designed. Primers identified by SEQ ID NO:1 and SEQ ID NO:2 of subsp. avenae reacted with all strains of A. avenae subsp. avenae (previously named P. avenae or P. alboprecipitans) originating from foxtail, oats, corn, rice, sugarcane, and millet, A. avenae subsp. cattleyae from orchid, and A. avenae subsp. citrulli (previously named P. pseudoalcaligenes subsp. citrulli) from watermelon. Primers identified by SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 of subsp. citrulli reacted with all strains of A. avenae subsp. citrulli, but not with any other strain of subsp. avenae. None of fifty-three other bacteria tested reacted with either set of primers. The citrulli- specific primers should prove especially useful for specific, sensitive, and rapid detection of this serious seedborne pathogen of watermelon seeds.

摘要翻译： 我们对代表来自几种宿主（包括狗尾草，燕麦，玉米，水稻，粟，甘蔗，兰花和西瓜）和西瓜的菌株的Acidovorax avenae菌株的16S-23S rDNA的内部间隔区的625和617bp片段进行了测序 A.Avenae subsp。 柑橘对病毒分别为西瓜，目的是设计PCR引物进行鉴定。 这些植物病原体以前被认为是非荧光假单胞菌，并且最近被重新分类为Acidovorax avenae subsp。 燕麦，A.Avenae subsp。 cattleyae和A. avenae subsp。 柑橘 设计了几套引物。 由SEQ ID NO：1和SEQ ID NO：2鉴定的引物。 Avenae与A.Avenae亚种的所有菌株反应。 源自狗尾草，燕麦，玉米，水稻，甘蔗和粟的Avenae（以前命名为P.Avenae或P.Aprapplipitans），A.Avenae subsp。 来自兰花的牛科，以及A.Avenae subsp。 柑橘（以前称为假产碱假单胞菌亚种柠檬酸）来自西瓜。 通过SEQ ID NO：3，SEQ ID NO：4，SEQ ID NO：5和SEQ ID NO：6鉴定的引物。 柑橘与所有A.Avenae亚种菌株反应。 柑橘，但不与任何其他亚种菌株。 燕麦 所检测的其他53种细菌均未与两组引物反应。 柑橘特异性引物应特别适用于西瓜种子严重的种子病原体的特异性，敏感和快速检测。

公开(公告)号：US06423499B1

公开(公告)日：2002-07-23

申请号：US09703807

申请日：2000-11-02

申请人： Wan-Yeob Song ,  Hyung-Moo Kim ,  Norman W. Schaad

发明人： Wan-Yeob Song ,  Hyung-Moo Kim ,  Norman W. Schaad

IPC分类号： C12Q168

CPC分类号： C12Q1/689 ,  C12Q2561/101

摘要： We sequenced a 619 and 617 bp fragment of the inner spacer region of 16S-23S rDNA of a strain of Acidovorax avenae representing pathogens from several hosts, including foxtail, oats, corn, rice, millet, sugarcane, orchid, and watermelon and a strain of A. avenae subsp. citrulli pathogenic only to watermelon and melons, respectively, for the purpose of designing PCR primers for their identification. These plant pathogens were previously considered as non-fluorescent pseudomonads and have been recently reclassified as Acidovorax avenae subsp. avenae, A. avenae subsp. cattleyae, and A. avenae subsp. citrulli. Several sets of primers were designed. Primers identified by SEQ ID NO:1 and SEQ ID NO:2 of A. avenae subsp. avenae reacted with all strains of A. avenae subsp. avenae (previously named P. avenae or P. alboprecipitans) originating from foxtail, oats, corn, rice, sugarcane, and millet, A. avenae subsp. cattleyae (previously named P. pseudoalcaligenes subsp. cattleyae) from orchid, and A. avenae subsp. citrulli (previously named P. pseudoalcaligenes subsp. citrulli) from watermelon and melon. Primers identified by SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 of subsp. citrulli reacted with all strains of A. avenae subsp. citrulli, but not with any other strain of A. avenae subsp. avenae. Primers identified by SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, reacted with the rice strains of A. avenae subsp. avenae, but not with any other strain of A. avenae subsp. avenae or any other subspecies of A. avenae. None of fifty-three other bacteria tested reacted with these sets of primers. The citrulli-specific primers and rice strain-specific primers should prove especially useful for the specific, sensitive, and rapid detection of these serious seedborne pathogens in watermelon and rice seeds, respectively.

公开(公告)号：US06410223B1

公开(公告)日：2002-06-25

申请号：US08334085

申请日：1994-11-04

申请人： Norman W. Schaad ,  Nikolas J. Panopoulos ,  Efstathios Hatziloukas

发明人： Norman W. Schaad ,  Nikolas J. Panopoulos ,  Efstathios Hatziloukas

IPC分类号： C12Q168

CPC分类号： C12Q1/6888 ,  C12Q1/686 ,  C12Q1/689

摘要： A novel polymerase chain reaction (PCR) technique which can specifically detect viable cells of a target cell or microorganism has been developed. The method combines a biological preamplification on growth medium with direct PCR and eliminates DNA extraction steps required for conventional PCR methods.

摘要翻译： 已经开发出可以特异性检测靶细胞或微生物的活细胞的新型聚合酶链反应（PCR）技术。 该方法将生物培养基上的生物预扩增与直接PCR结合，并消除常规PCR方法所需的DNA提取步骤。

公开(公告)号：US07262010B2

公开(公告)日：2007-08-28

申请号：US10283346

申请日：2002-10-30

申请人： Norman W. Schaad ,  Phillip E. Gaush ,  Meric Ozakman

发明人： Norman W. Schaad ,  Phillip E. Gaush ,  Meric Ozakman

IPC分类号： C12Q1/68

CPC分类号： C12Q1/689

摘要： Ralstonia solanacearum, the causal agent of bacterial brown rot of potato, is often carried latently in seed potato tubers. Primers and a probe were designed for a real-time BIO-PCR assay technique for detecting potato tubers latently infected with R. Solanacearum. Using naturally infected potato tubers, as few as 20 cells/ml extract could be detected. Two of 14 naturally infected potato tubers with no disease symptoms were positive by the newly described real-time BIO-PCR (pre-enrichment on agar or in liquid medium) assay but not by direct real-time PCR.

摘要翻译： 马铃薯细菌褐腐病的致病菌Ralstonia solanacearum通常在种薯马铃薯块茎中潜伏。 引物和探针设计用于实时BIO-PCR测定技术，用于检测潜在感染R. Solanacearum的马铃薯块茎。 使用天然感染的马铃薯块茎，可以检测到少至20个细胞/ ml的提取物。 通过新描述的实时BIO-PCR（在琼脂或液体培养基中的预富集）测定而不是通过直接实时PCR，14个没有疾病症状的天然感染的马铃薯块茎中的两个是阳性的。