Loop-mediated isothermal amplification (LAMP) detection of Phytophthora hibernalis, P. syringae and P. cambivora

Abstract

Phytophthora hibernalis, P. syringae and P. cambivora are devastating quarantine pathogens to many economically important plants worldwide. They often co-infect the same plant, causing rots and leading to great loss in yield and even to the death of the plant. Conventionally, the detection of the three pathogens has been based on time-consuming and laborious morphological methods and, although PCR-based detection is much better, it is still thermal cycler- and electrophoresis-dependent. A high throughput, rapid, convenient and cost-effective detection is required for quarantine detection at the port of entry. In this study, based on the novel LAMP technique, three sets of four LAMP primers, PH-F3/PH-B3/PH-FIP/PH-BIP, PS-F3/PS-B3/PS-FIP/PS-BIP and PC-F3/PC-B3/PC-FIP/PC-BIP, designed on the sequences of enolase (Enol), internal transcribed spacers (ITS) and ras-like protein Ypt1 genes of P. hibernalis, P. syringae and P. cambivora, were designed. Through the optimization of the reaction system and conditions, a highly specific and extremely sensitive LAMP detection system was developed for P. hibernalis, P. syringae and P. cambivora. The LAMP products showed the typical DNA ladders upon agarose gel electrophoresis, the visual turbidity of the reaction solution and the white precipitate of byproduct magnesium pyrophosphate upon centrifugation were observed; the LAMP solution turned light green from orange upon the addition of SYBR Green I. No cross reactions with the other closely related Phytophthora species were found, whereas specificity and sensitivity were very high, for the detection of 0.02 fg of genomic DNA was more than 105 times higher than the corresponding conventional PCR. To the best of our knowledge, this represents the first detection of the three quarantine Phytophthora pathogens by LAMP, a technique with a great potential for the identification of other Phytophthora pathogens.