Multiplex LAMP for Simplified Monitoring Cryptosporidium and Giardia in Surface Water

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Abstract
A detailed investigation was conducted into the application of an isothermal nucleic acid amplification procedure, LAMP, in conjunction with components of conventional procedure to determine ability to simplify water processing 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-α 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 Importance: Pathogen identification by LAMP is well established as a sensitive and highly selective procedure, insensitive to interferences, permitting specific detection of target DNA in samples containing extraneous DNA. This combination of features was shown effective for identifying numbers of Cryptosporidium oocysts and Giardia cysts as few as 1 in unseparated particle assemblages subject only to freeze thaw cycles. Although LAMP has not been widely adopted for environmental monitoring, it has been previously demonstrated for Cryptosporidium and for Giardia in water and wastewater. Also, many clinical assays have demonstrated applicability to use where facilities and skills are limited. The simplified analysis sequence offers significant time and cost saving for routine monitoring of Cryptosporidium and Giardia in surface water samples.

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