A multiplex PCR assay for the detection of Cryptosporidium species and simultaneous differentiation of Cryptosporidium hominis, Cryptosporidium parvum in clinical stool samples

Cryptosporidium hominis and Cryptosporidium parvum are 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 detect Cryptosporidium spp. and differentiates C. hominis and C. parvum from 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 positive Cryptosporidium DNA template. Best result with three sets of primers that amplifies 436 bp for all Cryptosporidium spp., 577 bp for C. hominis and 287 bp for C. parvum. In addition, thirty-five positive and thirty-five negative Cryptosporidium stool 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% for Cryptosporidium spp., C. hominis, and C. parvum respectively 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 of Cystoisospora belli and two known DNA sample of Cyclospora cayetanensis, available in our laboratory from the clinical stool samples. A single species-specific mPCR product of C. hominis and C. parvum were sequenced and deposited in GenBank database with the accession no MT862538 and MT875168 respectively. The mPCR assay is developed which differentiates C. hominis, and C. parvum in 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. HighlightsO_LINovel mPCR assay can detect all Cryptosporidium species C_LIO_LIThe sensitivity of the mPCR were 100%, 92.9%, and 87.5% for the primers designed to detect Cryptosporidium genus, C. hominis and C. parvum species respectively. C_LIO_LINo cross reactivity detected with newly developed mPCR assuring 100% specificity. C_LIO_LIThe developed mPCR assay is a robust, specific, reproducible, rapid and cost-effective molecular assay for the diagnosis of cryptosporidiosis. C_LIO_LIAssay is useful in molecular diagnosis of cryptosporidiosis, especially in developing countries. C_LI