Development of an immunomagnetic bead separation-coupled quantitative PCR method for rapid and sensitive detection of Cryptosporidium parvum oocysts in calf feces

Abstract

Cattle feces are the environmental vehicle for the zoonotic Cryptosporidium oocysts, but there are drawbacks associated with reliability of the existing methods for the detection of oocysts in the feces. Quantification of the immunomagnetic bead separation (IMS) coupled with real-time TaqMan PCR (qPCR) was accomplished by comparing the fluorescence signals obtained from the calf fecal samples of Cryptosporidium parvum oocysts with those obtained from standard dilutions of C. parvum oocysts. TaqMan qPCR assays were developed for the detection of C. parvum based on 18S rDNA gene. This IMS-qPCR assay allowed a reliable quantification of C. parvum oocysts over seven orders of magnitude with a baseline sensitivity of 8.7 oocysts. The newly developed IMS-qPCR technique proved specific as confirmed by negative reactivity against a wide panel of non-parvum Cryptosporidium oocysts. As a field application, experimentally infected calves (15 infected and 9 non-infected) were screened for oocysts shedding on 16, 18, and 21 days postinfection. Acid-fast staining microscopy of infected calves revealed oocysts in the feces of 11, 7, and 4 calves, respectively, compared to 15, 15, and 12 in case of screening by IMS-qPCR. Taken together, the proposed IMS-qPCR method significantly improved the diagnostic capacity for C. parvum infection in calves, making the technique a useful, sensitive, reliable, and time-saving.

Fast and Highly Sensitive Detection of Pathogens Wreathed with Magnetic Nanoparticles Using Dark-Field Microscopy

“…IMS combined with fluorescent microscropy method (named Method 1623, the C. parvum detection standard by American EPA) or IMS combined with PCR , both offer an accurate and reliable detection of C. parvum.…”

“…On the other hand, IMS technology has been applied extensively in separating and diagnosing various types of microorganisms, including bacteria, 13−15 viruses, 16,24 and parasites 17,18 from environmental samples due to its prominent advantages such as rapidity, low cost, and effectiveness. IMS combined with fluorescent microscropy method 25 (named Method 1623, the C. parvum detection standard by American EPA) or IMS combined with PCR 26,27 both offer an accurate and reliable detection of C. parvum. The limit of detection (LOD) of IMScoupled quantitative PCR method is 8.7 oocysts.…”

Ocorrência e identificação de Cryptosporidium e Giardia em amostras de água superficial destinada ao abastecimento público do estado de São Paulo

“…A técnica de raspagem de lâminas empregada para esse estudo foi adaptada a partir dos protocolos desenvolvidos por Nichols et al (2010) A introdução de PCR em tempo real com sondas de detecção fluorescente pode reduzir o risco de contaminação, tempo de trabalho e custos de reagentes através da possibilidade de combinar ensaios para a detecção de diferentes alvos em um ensaio (VERWEIJ et al, 2004). GAO et al, (2014) utilizaram separação imunomagnética (IMS) em conjunto com qPCR para melhorar a taxa de detecção de oocistos de Cryptosporidium parvum em fezes de vitelos. Os autores concluíram que os ensaios de detecção e quantificação são confiáveis para Cryptosporidium parvum a partir de amostras contaminadas, podendo ser aplicável a amostras ambientais, portanto, concluíram, que a técnica mostrou-se específica, sensível e eficiente.…”