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Development and evaluation of semi-nested PCR for detection of the variable lipoprotein haemagglutinin (vlhA) gene of Mycoplasma Synoviae in chicken

대한수의학회지 2020년 60권 3호 p.109 ~ 116
Pohuang Tawatchai, Phuektes Patchara, Junnu Sucheeva,
소속 상세정보
 ( Pohuang Tawatchai ) - Khon Kaen University Faculty of Veterinary Medicine
 ( Phuektes Patchara ) - Khon Kaen University Faculty of Veterinary Medicine
 ( Junnu Sucheeva ) - Khon Kaen University Faculty of Veterinary Medicine

Abstract


This study aimed to develop a semi-nested polymerase chain reaction assay for the direct detection of Mycoplasma synoviae (M. synoviae) from clinical samples using three newly designed oligonucleotide primers specific to the variable lipoprotein haemagglutinin (vlhA) gene and differentiate M. synoviae field strains based on a nucleotide deletion or the insertion of the prolinerich repeat (PRR) region of the vlhA gene. The developed semi-nested polymerase chain reaction (PCR) assay revealed positive results in 12 out of 100 clinical samples collected from chickens showing lameness and joint swelling. Six positive samples were selected randomly for sequencing, and sequence analysis revealed 96.3-100% nucleotide identities compared to the reference sequences. Phylogenetic analysis showed that sequences of the strains in this study were closely related to WVU1853 (Spain), CK.MS.UDL.PK.2014.2 (Pakistan), and F10-2AS (USA) strains, but they were distinct from the M. synoviae-H vaccine strain sequence. M. synoviae obtained from these samples were identified as types A and C with a length of 38 and 32 amino acids, respectively. These results indicated that the specific and sensitive semi-nested PCR could be a useful diagnostic tool for the direct identification of clinical samples, and the sequence analysis of the partial vlhA gene can be useful for typing M. Synoviae.

키워드

chicken; Mycoplasma synoviae; semi-nested PCR; vlhA gene

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