A multiplex PCR assay for the detection ofCryptosporidiumspecies and simultaneous differentiation ofCryptosporidium hominis, Cryptosporidium parvumin clinical stool samples

Abstract

AbstractCryptosporidium hominisandCryptosporidium parvumare responsible for more than 90% of the global cryptosporidiosis. Species identification is done by amplification of small subunit ribosomal ribonucleic acid (SSU rRNA) gene, followed by sequencing. We have developed a multiplex polymerase chain reaction (mPCR) assay which detectCryptosporidiumspp. and differentiatesC. hominisandC. parvumfrom stool samples without the need of post amplification sequencing. Nine new set of primers for mPCR assay were designed and the mPCR assay was standardized with known positiveCryptosporidiumDNA template. Best result with three sets of primers that amplifies 436 bp for allCryptosporidiumspp., 577 bp forC. hominisand 287 bp forC. parvum. In addition, thirty-five positive and thirty-five negativeCryptosporidiumstool samples identified by the gold standard nested 18S rRNA PCR-sequencing assay were tested by mPCR. The sensitivity of the mPCR are 100%, 92.9%, and 87.5% forCryptosporidiumspp.,C. hominis, andC. parvumrespectively while specificity is 100% for all the three primers. No cross-reactivity was observed by the new mPCR assay when tested with five known DNA sample ofCystoisospora belliand two known DNA sample ofCyclospora cayetanensis, available in our laboratory from the clinical stool samples. A single species-specific mPCR product ofC. hominisandC. parvumwere sequenced and deposited in GenBank database with the accession noMT862538andMT875168respectively. The mPCR assay is developed which differentiatesC. hominis, andC. parvumin a single test run of amplification and without the need for RFLP or sequencing. Although it less sensitive than 18S rRNA PCR-sequencing assay, but 100% specific, rapid, cost-effective and suitable for making diagnosis of cryptosporidiosis especially in developing countries.HighlightsNovel mPCR assay can detect allCryptosporidiumspeciesThe sensitivity of the mPCR were 100%, 92.9%, and 87.5% for the primers designed to detectCryptosporidiumgenus,C. hominisandC. parvumspecies respectively.No cross reactivity detected with newly developed mPCR assuring 100% specificity.The developed mPCR assay is a robust, specific, reproducible, rapid and cost-effective molecular assay for the diagnosis of cryptosporidiosis.Assay is useful in molecular diagnosis of cryptosporidiosis, especially in developing countries.