Development of loop-mediated isothermal amplification (LAMP) assay combined with malachite green as a rapid screening test for Candidatus Mycoplasma haemominutum infection in cats

Abstract

Feline infectious anaemia is caused by a Gram-negative, uncultivable, cell wall-deficient, epierythrocytic parasitic bacteria known as feline haemoplasmas (FHM) in the genus Mycoplasma, namely, Mycoplasma haemofelis (Mhf), Candidatus M. haemominutum (CMhm), and Ca. M. turicensis (CMtc). Here, a loop-mediated isothermal amplification (LAMP) targeting the 16S rRNA gene combined with malachite green (MG) based colorimetric assay was developed for the detection of feline CMhm infection. The limit of detection was determined using a ten-fold serial dilution of recombinant plasmid DNA (from 108 to 1 copies). The result indicated that the LAMP-MG assay could detect as low as 264 copies corresponding to 264 organisms/μl of CMhm feline blood. Comparison between the LAMP-MG and standard polymerase chain reaction (PCR) surveying 105 clinical samples suggested that 17 and 15 samples were positive for CMhm, respectively. Validity of the LAMP-MG assay was assessed and calculated within 95% confidence intervals (CIs). The sensitivity, specificity, prevalence and accuracy were 100.0%, 97.8%, 14.3%, and 98.1%, respectively. The degree of agreement between LAMP-MG and standard PCR assays was 92.6%, with a κ coefficient of 1 (CI: 82.5-100.0%). This LAMP-MG colorimetric assay may be applicable as a rapid screening point-ofcare testing for feline CMhm, as well as for blood donors prior to blood transfusion by using unsophisticated equipment, such as a heating block or water bath. This technique could provide robust results, which are easily distinguished within 60 min after amplification. © 2017 Science Society of Thailand under Royal Patronage. All Rights Reserved.