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Development and application of a universal Hemoplasma screening assay based on the SYBR green PCR principle
Barbara Willi1, Marina L Meli, Ruedi Lüthy
1Clinical Laboratory, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland.
Abstract:
Hemotropic mycoplasmas (hemoplasmas) are the causative agents of infectious anemia in several mammalian species. Their zoonotic potential has recently been substantiated by the identification of a feline hemoplasma isolate in an immunocompromised human patient. Although species-specific diagnostic molecular methods have been developed, their application as screening tools is limited due to the species diversity of hemoplasmas. The goals of this study were to develop a universal hemoplasma screening assay with broad specificity based on the SYBR green PCR principle, to compare the assay with hemoplasma-specific TaqMan PCR, and to analyze potential tick vectors and human blood samples to address the zoonotic potential. The newly developed PCR assay based on the 16S rRNA gene amplified feline, canine, bovine, porcine, camelid, and murine hemoplasmas, as well as Mycoplasma penetrans and Mycoplasma pneumoniae. The lower detection limit for feline and canine hemoplasmas was 1 to 10 copies/PCR. The assay exhibited 98.2% diagnostic sensitivity and 92.1% diagnostic specificity for feline hemoplasmas. All 1,950 Ixodes ticks were PCR negative, suggesting that Ixodes ticks are not relevant vectors for the above-mentioned hemoplasma species in Switzerland. None of the 414 blood samples derived from anemic or immunocompromised human patients revealed a clear positive result. The SYBR green PCR assay described here is a suitable tool to screen for known and so-far-undiscovered hemoplasma species. Positive results should be confirmed by specific TaqMan PCR or sequencing.
Insights
A new SYBR green PCR assay effectively screens for diverse hemotropic mycoplasmas (hemoplasmas) in mammals. This broad-spectrum test aids in detecting known and novel hemoplasma species, crucial for understanding infectious anemia and zoonotic potential.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Zoonotic Disease Research
Background:
- Hemotropic mycoplasmas (hemoplasmas) cause infectious anemia in mammals.
- Zoonotic transmission of hemoplasmas is a growing concern, highlighted by a feline isolate in an immunocompromised patient.
- Existing species-specific PCR assays are limited for broad hemoplasma screening due to species diversity.
Purpose of the Study:
- Develop a universal SYBR green PCR assay for broad-spectrum hemoplasma screening.
- Compare the new assay's performance against specific TaqMan PCR methods.
- Investigate potential tick vectors and human blood samples for zoonotic hemoplasma transmission.
Main Methods:
- Developed a SYBR green PCR assay targeting the 16S rRNA gene for universal hemoplasma detection.
- Validated the assay using various mammalian hemoplasma species, Mycoplasma penetrans, and Mycoplasma pneumoniae.
- Compared diagnostic sensitivity and specificity with hemoplasma-specific TaqMan PCR.
- Analyzed Ixodes tick samples and human blood from immunocompromised patients.
Main Results:
- The SYBR green PCR assay successfully amplified feline, canine, bovine, porcine, camelid, and murine hemoplasmas, plus M. penetrans and M. pneumoniae.
- Achieved a low detection limit of 1-10 copies/PCR for feline and canine hemoplasmas.
- Demonstrated high diagnostic sensitivity (98.2%) and specificity (92.1%) for feline hemoplasmas.
- Found no evidence of hemoplasmas in tested Ixodes ticks in Switzerland.
- No positive results were obtained from human blood samples.
Conclusions:
- The developed SYBR green PCR assay is a valuable tool for screening known and novel hemoplasma species.
- The assay aids in diagnosing hemoplasma infections and assessing zoonotic risks.
- Ixodes ticks are unlikely vectors for these hemoplasma species in the studied region.
- Further confirmation of positive SYBR green PCR results using TaqMan PCR or sequencing is recommended.
