Table 2. Sequences of primers, probes, PCR product sizes and amplification efficiency of the qPCR assays^a.

Organism	Target	Primer and probeb	Primer and probe sequence (5′ to 3′)	nM	Product (bp)	qPCR efficiency (%)c
						Singleplex	Multiplex
Mycoplasma sppd	16S rRNA	Myco_F Myco_R Myco_Probe	CGAGCGCAACCCTTATCCTT CCCCACTCGTAAGAGGCATGA VIC-TCGTCCCCACCTTCCTCCCG-QSY	100 100 75	118	88.8–100.9	96.8–102.4
M. bovis	rpoB	M.bovis_F M.bovis_R M.bovis_Probe	TTTCAGCCGCTAACTTCAGAGC GCAAGTTCCCCATCCTTGAAG ABY-TCGCCTTTAGCAACTTCTTGACCAA-QSY	200 200 200	232	87.1	95.6
A. laidlawii	ITS	Achol_F Achol_R Achol_Probe	AAGTGGGCAATACCCAACGC ACGTTCCCGTAGGGATACCTTG 6-FAM-ACGGCTCCCTCCCTTTCGGG-QSY	15015075	108	91.9	91.2

Notes:

^aThe QuantiFast Multiplex PCR master mix (Qiagen, Redwood City, CA, USA) was used for all assays and with an annealing temperature of 58 °C.

^bF and R indicate forward and reverse primers, respectively. TaqMan probes were designed with 6-FAM (6-carboxyfluorescein) VIC (2’-chloro-7phenyl-1,4-dichloro-6-carboxy-fluorescein) and ABY (Thermo Fisher, Waltham, MA, USA) as reporter dyes on the 5′ end and QSY 7 (QSY7 succinimidyl ester) as the quencher dye on the 3′ end.

^cAmplification efficiencies were determined using gDNA from M. bovis ATCC25523, M. californicum ATCC 33461, and M. bovigenitalium ATCC19852 (16S rRNA), M. bovis ATCC25523 (rpoB), and A. laidlawii ATCC 23206 (ITS).

^dThe PCR amplification efficiencies for M. bovis, M. californicum ATCC 33461, and M. bovigenitalium ATCC19852 were measured as 88.8%, 97.1%, and 100.9% in singleplex and 96.8, 102.4% and 97.9% in multiplex, respectively.