In comparison with the results of conventional single PCR, the sensitivities of the GeXP assay for detection of aac(3)-II, aac(6′)-Ib, aac(6′)-II, ant(3″)-I, aph(3′)-VI, armA and rmtB were 92.50% (37/40), 100% (11/11), 88.89% (8/9), 100% (40/40), find more 83.33% (10/12), 95.24% (40/42) and 93.33% (14/15),respectively, and the specificities were 88.23% (15/17), 93.33% (42/45), 95.74% (45/47), 87.50% (14/16), 100% (44/44), 85.71% (12/14)

and 92.68% (38/41), respectively. The Kappa values of the GeXP assay and conventional single PCR for detecting the seven resistance genes were 0.831, 0.846, 0.810, 0.909, 0.887, 0.810 and 0.825, respectively. Those specimens that were negative by the conventional single PCR but Caspase activity assay Positive by the GeXP assay (Table 5) were confirmed later by mono-GeXP assay and sequenced to be true positives, suggesting the optimized GeXP assay performed a better sensitivity than the conventional method. Meanwhile, some genes were detected as positive by conventional method but negative

by multiplex GeXP assay (4th column of Table 5). The false negative genes of multiplex GeXP assay could be detected by mono-GeXP assay with less than 2000 A. U. dye signal strength of the peaks CT99021 mouse in these false negatives. The later analysis of the distribution of seven aminoglycoside-resistance genes showed that all of false negatives of multiplex GeXP assay harbored more than four genes, and the concentration of each gene in these isolates largely varied, suggesting the false negatives of multiplex GeXP assay were missed due to the ignorance of the lower peak (less than 2000 A. U. dye signal strength) overcast by higher peaks (more than 100000 A. U.). Table 5 Comparison of GeXP assay and conventional single PCR for detecting seven aminoglycoside-resistance genes CHIR-99021 molecular weight Resistance genes GeXP assay Conventional single PCR Measures of agreement Positive Negative Total Kappa values* aac(3)-II Positive 37 2 39 0.831 (P=0.000) Negative 2 15 17 Total 39 17 56 aac(6′)-Ib Positive 11 3 14 0.846 (P=0.000) Negative

0 42 42 Total 11 45 56 aac(6′)-II Positive 8 2 10 0.810 (P=0.000) Negative 1 45 46 Total 9 47 56 ant(3″)-I Positive 40 2 42 0.909 (P=0.000) Negative 0 14 14 Total 40 16 56 aph(3′)-VI Positive 10 0 10 0.887 (P=0.000) Negative 2 44 46 Total 12 44 56 armA Positive 40 2 42 0.810 (P=0.000) Negative 2 12 14 Total 42 14 56 rmtB Positive 14 3 17 0.825 (P=0.000) Negative 1 38 39 Total 15 41 56 *As a rule of thumb, values of Kappa from 0.40 to 0.59 are considered moderate, 0.60 to 0.79 substantial, and 0.80 outstanding. The GeXP assay in our study can simultaneously detect 7 genes related to resistance to aminoglycosides. The cost is about 5£ per test, versus 5£ using commercial real-time PCR kit for individual gene in a single PCR assay.