A new immunomagnetic bead separation–surfactant extraction treatment protocol for rapid and sensitive quantitative PCR detection of Cryptosporidium parvum DNA

Takahiro Sekikawa

doi:10.2166/ws.2016.125

Abstract

The Cryptosporidium oocyst is encased in a robust wall that is extremely resistant to detrimental environmental factors such as chlorine used to disinfect potable water. Therefore, extracting oocyst DNA is not a trivial undertaking. Standard procedures used to extract DNA from oocysts, such as freeze–thaw (F/T) methods and DNA purification kits, are time-consuming and expensive and are difficult to implement in routine clinical practice. Therefore, we developed a surfactant extraction treatment (SET) that efficiently extracts DNA from the oocyst. Immunomagnetic separation (IMS) combined with quantitative real-time polymerase chain reaction (qPCR) detects pathogenic microorganisms with high sensitivity. The objective of the present study was to evaluate SET for its ability to simplify qPCR detection of 18S rDNA directly from immunomagnetic bead–oocyst conjugates. DNA extracted directly from the conjugates using SET did not affect DNA amplification in the qPCR assay. Further, the rate of DNA amplification using IMS–SET was greater than that using F/T combined with the DNA purification kit. The rate of recovery of oocysts from surface water samples spiked with oocysts did not differ significantly from previously published values. These data demonstrate that the new IMS–SET protocol using qPCR can simplify the recovery and detection of Cryptosporidium oocysts.

Highlights

Cryptosporidium parvum was first recognized as a human pathogen in 1976 (Nime et al ), nearly 70 years after the first cryptosporidial species was identified in mice (Tyzzer )
Cryptosporidium infects various vertebrate hosts, including mammals, rodents, birds, reptiles, and fish; and oocysts excreted by these hosts are present in the environment (Smith & Nichols )
The link with AIDS was so strong that cryptosporidiosis became one of the defining features of the syndrome before the discovery of the human immunodeficiency virus (HIV) (Tzipori & Widmer )

Summary

Introduction

Cryptosporidium parvum was first recognized as a human pathogen in 1976 (Nime et al ), nearly 70 years after the first cryptosporidial species was identified in mice (Tyzzer ). Cryptosporidium is a significant pathogen in all populations regardless of the prevalence of HIV. It is the second most common pathogen in infants (Kotloff et al ).

Objectives

Methods

Results