Multiplex LAMP for Simplified Monitoring Cryptosporidium and Giardia in Surface Water

Isothermal nucleic acid amplification, LAMP, was applied with components of conventional procedure for simplified water processing to assess its value for monitoring Cryptosporidium and Giardia in surface water samples. Based on previous work demonstrating a high degree of sensitivity and selectivity of the LAMP procedure for detecting target organism DNA at low concentration in environmental media without significant interferences, this project sought to determine if LAMP primers for both organisms could be combined and applied to detect the target organisms collected from surface water samples after only minimal processing. Implementation consisted of optimizing previously described LAMPs for the SAM-1 gene of Cryptosporidium and for the EF1-a gene of Giardia; establishing their limits of detection on both specific DNA and on small numbers of oocysts and cysts processed by freeze-thaw cycles. Based on positive results from preliminary testing, a multiplexed LAMP for both organisms was applied. The multiplex lamp was able to detect the DNA of Cryptosporidium and of Giardia from samples seeded with replicates of 1, 2, 3 and 4 oocysts and cysts, both in clean water and in surface water processed by filtration and centrifuging to pellet followed only by freeze-thaw cycles. Positive and specific amplification was observed with potential for quantification using 25 µL reactions including 8 µL template, in RT qLAMP format by LightCycler. The procedure is significantly simpler and less time consuming presenting realistically practical procedure for improvement of surface water monitoring of Cryptosporidium and Giardia, compared to the widely used USEPA Method 1623.