MLVA typing
MLVA typing was completed based on a culture-independent method by Chao
Yan et al. with slight modifications.13 Briefly, PCR
was done using primers targeting the four loci containing tandem repeat
sequence (Table 1). The PCR was completed on a Verity thermal cycler
(Thermo Fisher Scientific Inc) following this methodology: step 1, 94°C
for 10 min; step 2, 94°C for 1min, 58°C for 1min, and 72°C for 1min and
30 s. The second step was iterated for 35 cycles. The label PCR products
were separated by capillary electrophoresis using an 3500DX Genetic
Analyzer platform (Thermo Fisher Scientific, USA),and the data were
analyzed using GeneMapper software (version 5.0;Applied Biosystems).