Loop-Mediated Isothermal Amplification Assay for the Detection of Plant Pathogens
Author: A. Kandan, J. Akhtar, Pardeep Kumar and Z. Khan
Division of Plant Quarantine, ICAR-National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi - 110012
Nucleic acid amplification is a valuable tool for diagnosis of plant pathogens at the early stage of infection. Early diagnosis of plant pathogens may leads to quick management of such a pathogen infection and reduces the crop yield loss to the growers. Loop-mediated isothermal amplification (LAMP) offers as a field oriented and user friendly alternative to polymerase chain reaction (PCR). LAMP is less time intensive than PCR and can be performed using heat-blocks, with results read by eye under UV light. LAMP is a very simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR technique, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification.
Principle of LAMP
The fundamental amplification reaction requires four types of primers which are complementary to six regions of the target gene. As double stranded DNA of plant pathogens is in a condition of dynamic equilibrium at a temperature around 63°C - 65°C. One of the LAMP primers can anneal to the complementary sequence of double stranded target DNA of the pathogen and further initiating DNA synthesis with the DNA polymerase with strand displacement activity displacing and releasing a single stranded DNA. Next, due to the complementarity of the 5' end of the forward inner primer (FIP) and backward inner primer (BIP) in nearby regions of the target amplicon, loop structures are formed. This further allows various sized structures consisting of alternately inverted repeats of the target DNA sequence on the same strand to be formed in rapid succession. The addition of loop primers, which contain sequences complementary to the single stranded loop region on the 5' end of the hairpin structure, eventually speeds the reaction by providing a greater number of starting points for DNA synthesis. Using loop primers, amplification by 109-1010 times can be achieved within 15-30 minutes. Use of loop primers is not mandatory in LAMP reaction.
The LAMP assay is performed under isothermal conditions, employing a Bst DNA polymerase with principle of strand-displacing activity involving a set of four specially designed primers which recognize a total of six distinct sequences on the target DNA to be amplified. The amplified products contain single-stranded loops, allowing primers to bind without the need for repeated cycles of thermal denaturation. LAMP products can be visualized with the unaided eye by adding DNA intercalating dyes such as ethidium bromide, propidium iodide, SYBR Green I, or Quant-iT PicoGreen; by adding metal-ion indicators such as hydroxynaphthol blue (HNB)., CuSO4, calcein, or by measuring the increase in turbidity derived from magnesium pyrophosphate formation. LAMP products can also be visualized as a ladder-like banding pattern on 2% gel electrophoresis.
Application of LAMP
Positive LAMP reaction can also be monitored in real-time, allowing quantitative detection of the target. LAMP is relatively insensitive to the accumulation of DNA and DNA by-products (pyrophosphate salts), so the reaction proceeds until large amounts of amplicon are generated. There is no need of special equipment to do LAMP assay. The LAMP method can amplify DNA with high specificity and efficiency without the need for special equipment, it is suitable for the detection and identification of plant pathogens at agriculture sites. When compared with standard PCR, the LAMP assay reported here has the advantages of simple detection and rapid assay time (< 70 min). A thermal cycler is not required because there is no heat denaturation step, and a regular laboratory water bath or a heating block that can provide a constant temperature (65°C) can be used. As the LAMP reaction progresses, pyrophosphate ions are produced; these bind to Mg2+ ions and form a white precipitate of magnesium pyrophosphate. Therefore, the results of the LAMP can be judged by the unaided eye. This characteristic feature of the LAMP reaction means that the reaction end point can be detected simply by gauging the presence of a precipitate.
LAMP tests haven been developed for rapid detection of few fungi viz. Sclerotinia sclerotiorum, Synchrticum endobioticum, Pythium helicoides, Phytophthora sojae, Phytophthora ramorum, Fusarium oxysporum, Fusarium equiseti, Fusarium graminearum, few bacteria viz. Erwinia amylovora, Xanthomonas citri subsp. citri, Xylella fastidiosa and few viruses like banana streak virus and nematode namely Meloidogyne enterolobii.
Other than plant pathogens, LAMP assay also developed to detect the foodborne pathogens and few toxicants. LAMP developed to detect few foodborne pathogens which are as follows: Salmonella typhimurium, Salmonella enterica, Escherichia coli, Vibrio vulnificus, Vibrio cholerae, Yersinia enterocolitica, Cronobacter sakazakii, Shigella spp., Shewanella putrefaciens, Burkholderia pseudomallei, Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, and few toxicants like Aspergillus flavus, Aspergillus nomius, Aspergillus parasiticus, Aspergillus carbonarius, Dekkera anomala and Dekkera bruxellensis.
About Author / Additional Info:
Senior Scientist (Plant Pathology), Division of Plant Quarantine, Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi-110012.