sybr-green-i and Cat-Diseases

Topics: Animals; Astroviridae; Benzothiazoles; Cat Diseases; Cats; Diamines; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Sensitivity and Specificity

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.

Topics: Animals; Benzothiazoles; Cat Diseases; Cats; Cattle; Diamines; DNA, Bacterial; Dogs; Humans; Mass Screening; Mice; Mycoplasma; Mycoplasma Infections; Organic Chemicals; Polymerase Chain Reaction; Quinolines; RNA, Ribosomal, 16S; Sensitivity and Specificity; Sequence Analysis, DNA; Switzerland; Taq Polymerase

This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) assay with SYBR Green targeting the VP2 (ORF 3) of feline caliciviruses. All of the 44 feline calicivirus isolates tested were detected, whereas neither feline herpesvirus, feline panleukopenia virus, feline immunodeficiency virus and feline leukemia virus nor other calicivirus like rabbit hemorrhagic disease virus and a canine calicivirus isolate showed specific amplification products. The sensitivity of the SYBR Green reaction was shown to be equivalent to 5 x 10(1) to 5 x 10(2) copies/reaction and the overall sensitivity equivalent to a feline calicivirus titer of 10(0.6) TCID(50)/100 microl in Crandell Reese Feline Kidney (CRFK) cells.

Topics: Animals; Benzothiazoles; Caliciviridae Infections; Calicivirus, Feline; Cat Diseases; Cats; Cell Line; Diamines; Fluorescent Dyes; Nucleic Acid Denaturation; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sensitivity and Specificity; Temperature; Templates, Genetic