Rapid-cycle PCR detection of Pyrenophora graminea from barley seed

被引：34

作者：

Taylor, EJA ^{[1
]}

Stevens, EA ^{[1
]}

Bates, JA ^{[1
]}

Morreale, G ^{[1
]}

Lee, D ^{[1
]}

Kenyon, DM ^{[1
]}

Thomas, JE ^{[1
]}

机构：

[1] Natl Inst Agr Bot, Cambridge CB3 0LE, England

来源：

PLANT PATHOLOGY | 2001年 / 50卷 / 03期

关键词：

Pyrenophora graminea; rapid PCR detection; SCAR; seed health test;

D O I：

10.1046/j.1365-3059.2001.00563.x

中图分类号：

S3 [农学（农艺学）];

学科分类号：

0901 ;

摘要：

Sixty RAPD primers were used to screen for a diagnostic marker that could be used to identify Pyrenophora graminea, a fungal seedborne pathogen that causes leaf stripe on barley. Primer pairs were designed to differentiate P. graminea from other Pyrenophora spp. using a sequence-characterized amplified region (SCAR) approach. A pair of P. graminea-specific primers (PG2 F/R) was obtained that amplified a single fragment from 37 isolates of P. graminea tested, but not from 29 isolates of other Pyrenophora spp. or 12 saprophytes isolated from barley seed. Rapid PCR detection was achieved using a LightCycler, in which the emission of fluorescence from the binding of SYBR Green I dye to the PCR products is measured. The P. graminea-specific product resulting from amplification with PG2 FIR can be distinguished from any nonspecific products by post-PCR melting point analysis. The PCR assay involves 40 amplification cycles of PCR, and the total PCR test including melting point analysis takes 25 min to complete. The rapidity of this test, combined with the closed 'in-tube' detection of PCR products, which reduces the potential for contamination, offers significant advantages compared with conventional laboratory and PCR analyses.

引用

页码：347 / 355

页数：9

共 25 条

[1] THE MOLECULAR DIVERSITY OF THE 5S RIBOSOMAL-RNA GENE IN BARLEY (HORDEUM-VULGARE) [J].