Detection and identification of Giardia species using real-time PCR and sequencing

N Jothikumar; Jennifer L Murphy; Vincent R Hill

doi:10.1016/j.mimet.2021.106279

Detection and identification of Giardia species using real-time PCR and sequencing

J Microbiol Methods. 2021 Oct:189:106279. doi: 10.1016/j.mimet.2021.106279. Epub 2021 Jul 14.

Authors

N Jothikumar¹, Jennifer L Murphy², Vincent R Hill³

Affiliations

¹ Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Waterborne Disease Prevention Branch, Atlanta, GA, USA. Electronic address: njothik@gmail.com.
² Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Waterborne Disease Prevention Branch, Atlanta, GA, USA. Electronic address: JLMurphy@cdc.gov.
³ Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Waterborne Disease Prevention Branch, Atlanta, GA, USA. Electronic address: veh2@cdc.gov.

PMID: 34271057
DOI: 10.1016/j.mimet.2021.106279

Abstract

We report a specific region of Giardia spp. 18S ribosomal RNA (18S rDNA) that serves as an ideal target for quantitative PCR (qPCR) detection and sequencing to identify Giardia species, including the clinically-relevant G. duodenalis, in clinical and environmental samples. The presence of multiple copies of the 18S rDNA gene and variations in the selected 18S genomic region enabled the development of a rapid, sensitive qPCR screening method for the detection of Giardia spp. The analytical sensitivity of the Giardia qPCR assay was determined to be a cyst equivalent of 0.4 G. duodenalis cysts per PCR reaction. Amplicon sequencing of the PCR product confirmed Giardia spp. detection and among the 35 sequences obtained, 31, 3 and 1 isolates were classified as belonging to G. duodenalis, G. microti and G. muris, respectively. The TaqMan assay reported here may be useful for the detection of low levels of Giardia in clinical and environmental samples, and further enables the effective use of direct sequencing of the PCR product for Giardia confirmation and to identify major species of Giardia, including G. duodenalis.

Keywords: Giardia; Sequencing; Zoonotic; qPCR.

MeSH terms

DNA, Ribosomal / genetics
Feces / parasitology
Genotype
Giardia / classification
Giardia / genetics*
Giardiasis / diagnosis
Giardiasis / parasitology
RNA, Ribosomal, 18S / genetics
Real-Time Polymerase Chain Reaction / methods*
Real-Time Polymerase Chain Reaction / standards
Sequence Analysis, DNA / methods*

Substances

DNA, Ribosomal
RNA, Ribosomal, 18S