Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

Abstract

BackgroundCitrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment.ResultsA loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA of other phytopathogenic bacteria. The assay was capable of detecting CBC-causing strains from several geographical origins and pathotypes.ConclusionsThe CBC-LAMP technique is a simple, fast, sensitive and specific method for the diagnosis of Citrus Bacterial Canker. This method can be useful in the phytosanitary programs of the citrus industry worldwide.

Sensitive Detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by Loop-Mediated Isothermal Amplification

“…Therefore, we predict that the inherent efficiency of annealing for each primer set causes this variation. Regardless, the sensitivity thresholds among the four assays developed were consistent with those previously reported for other plant-pathogenic bacteria, ranging from 10 fg to 0.01 ng genomic DNA and 10 3 to 10 4 CFU ml Ϫ1 (22,(35)(36)(37)42), and correlate with the equivalent range of 10 3 to 10 6 genome copies based on the 5.2-Mbp X. oryzae pv. oryzae PXO99…”

New Developments in Pathogen Detection and Identification

“…Although developed ten years ago, it started being intensively used in the medical fields three years ago (more than 200 references since than). In comparison the use of LAMP in phytodiagnostics is still incipient; nevertheless it has been applied for the detection of viruses, viroids, bacteria, fungi and nematodes (Fukuta et al 2003;Fukuta et al 2004;Nie 2005;Varga and James 2006;Tomlinson et al 2007;Kubota et al 2008;Boubourakas et al 2009;Kikuchi et al 2009;Kuan et al 2010;Rigano et al 2010;Zhao et al 2010). The original LAMP method relies in the activity of Bst DNA polymerase, a polymerase that has a strand displacement activity, and four primers which anneal to 6 regions of the template (two of the primers are specific for more than one region).…”

“…Depletion of magnesium ion due to precipitation as magnesium pyrophosphate can also be colorimetrically quantified with hidroxynaphtol blue, a reagent commonly used to titrate the amount of alkaline earth ions in water (Goto et al 2009). A lateral flow device has also been used for the detection of amplified products (Rigano et al 2010). However, some occasional reports of unspecific amplifications (see Curtis et al (2009)) may restrain the enthusiasm in the development of unspecific methods for monitoring the amplification.…”

Rapid detection of Colletotrichum gloeosporioides in infected strawberry plants using loop-mediated isothermal amplification

“…The LAMP assay has been used to detect pathogenic plant viruses (Fukuta et al 2003, 2004, 2013b), viroids (Boubourakas et al 2009), fungi (Duan et al 2014a, b;Fukuta et al 2014;Lenarčič et al 2014;Moradi et al 2014;Takahashi et al 2014;Tomlinson et al 2010;Zhang et al 2013), bacteria (Okuda et al 2005;Rigano et al 2010Rigano et al , 2014, oomycete pathogens (Dai et al 2012;Fukuta et al 2013a;Tomlinson et al 2007), and nematodes (Kikuchi et al 2009). The LAMP assay requires 4 different primers that recognize 6 distinct regions of a target gene.…”