High Throughput Multiplex PCR and Probe-based Detection with Luminex Beads for Seven Intestinal Parasites

Abstract

Abstract. Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and coproantigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites-Cryptosporidium spp., Giardia intestinalis , Entamoeba histolytica , Ancylostoma duodenale , Ascaris lumbricoides , Necator americanus , and Strongyloides stercoralis -were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites.

Recombinase Polymerase Amplification-Based Assay to Diagnose Giardia in Stool Samples

“…This is roughly equivalent to other PCRbased nucleic acid assays, which show sensitivities of 10 3 -10 4 Giardia cysts per milliliter of stool. 11,22,23 Figure 1. The recombinase polymerase amplification-based Giardia (RPAG) assay consistently detects as few as 10 3 -10 3.5 cysts per milliliter of stool during bench top testing.…”

“…These false negative RPAG assay results could have been caused by the slightly lower limit-of-detection of PCR assays compared with microscopy or the RPAG assay. 11 It is also possible that some of these samples were falsely positive by PCR. Seven of the 17 false negative RPAG results were positive by microscopy and PCR, suggesting that the limit of detection must by further improved.…”

“…A number of nucleic acid-based and antigen-based diagnostic assays for stool sample detection of Giardia at the point-of-care are available and have shown impressive reliability. [9][10][11] Traditional nucleic acid diagnostics such as polymerase chain reaction (PCR), however, require the use of expensive thermal cycling equipment, limiting their use to central laboratories.…”

Management of patients with suspected infectious diarrhoea in hospitals in England

“…These advances include multiplex polymerase chain reaction (PCR) assays and whole genome sequencing 3,4 . Prior to implementing these new tests, it is important to establish how patients with infectious diarrhoea are currently managed in hospitals in order to assess the implications of more widespread molecular testing.…”

Soil transmitted Helminth infestation in diarrhoeal and non-diarrhoeal children in Mirpur slum area of Dhaka city

“…Phuphisut et al (2014) also compared microscopic examination of fecal samples with molecular assay and revealed that sensitivity and specificity of the multiplex PCR was 87 and 83%, respectively. According to Taniuchi et al (2011), multiplex PCR-bead protocol which is applied in the present study, provides an alternative high-throughput molecular diagnostic platform for specific and sensitive detection and a potential alternative to microscopy for equipped laboratories.…”