Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing.

This study discusses the use of LAMP (loop-mediated isothermal DNA amplification) assays in detecting Cryptosporidium species DNA in feces from cattle, sheep, and horses in South Africa, producing higher detection results compared to nested PCR methods. The results from these LAMP assays were further validated through sequencing.

Understanding the Research

• The primary purpose of the research was to check the effectiveness of LAMP assays in detection of Cryptosporidium, in comparison to traditional nested PCR methods.
• The researchers utilized three types of LAMP assays: SAM-1, HSP, and gp60. These assays were used to detect three different kinds of Cryptosporidium.
• The study used 270 fecal samples collected from cattle, sheep, and horses in South Africa. The DNA for the test was extracted from 0.5 g of each fecal material.

Difference between LAMP and PCR Results

• All of the 270 samples tested negative in nested PCR assays. Conversely, up to a third of the same samples tested positive in the LAMP assays
• More specifically, the SAM-1 LAMP assay detected Cryptosporidium DNA in different proportions of cattle (33.64%), sheep (30.5%), and horses (21.79%) samples.
• The HSP LAMP test detected Cryptosporidium DNA in small proportions of cow (0.9%), sheep (5.8%), and horse (8.9%) samples.
• The gp60 LAMP test, which was designed to detect Cryptosporidium parvum, didn’t amplify any Cryptosporidium DNA, presumably due to low sample DNA concentrations.

Concluding the Research

• The study further validated the positive results from LAMP assays by sequencing the amplified DNA products. The sequences showed a high analogy with the target genes, confirming the accuracy of the LAMP assay results.
• The researchers concluded that LAMP assays were superior to nested PCR in detecting Cryptosporidium species DNA, making them an effective tool for epidemiological survey studies.
• The study recommends LAMP method particularly for screening healthy animals for Cryptosporidium, as these animals typically have low oocyst shedding, often going undetected by PCR in conventional sample concentrates.