Validation and application of an immunochromatographic test to detect four macrolides and two lincosamides in raw cow milk

In this study, an immunochromatographic test (using the Charm QUAD2® Test) was used to screen for residual macrolides and lincosamides in raw cow’s milk. The validation parameters (selectivity/specificity, detection capability (CCβ), and ruggedness) were in agreement with the requirements of[EC] 2021. The selectivity of the immunochromatographic test was verified by the negative results of microbiological tests. The false-positive rate was 0%. The CCβ values of the immunochromatographic test for various antibiotics in milk were as follows: erythromycin 0.02 mg/kg, spiramycin 0.1 mg/kg, tilmicosin 0.025 mg/kg, tylosin 0.05 mg/kg, lincomycin 0.15 mg/kg, and pirlimycin 0.15 mg/kg. The determined CCβ values were lower than the respective maximum residue limits (MRLs; regulatory limits in Japan) for milk, except for lincomycin (equal to the MRL). The presence of antibiotic groups other than macrolides and lincosamides did not interfere with the specificity of the test. It showed no significant difference in lot-to-lot repeatability. The results obtained by the two researchers showed no significant differences. Finally, the test was applied to milk samples obtained from a tylosin-treated cow. The outcome was positive and in agreement with the results of the chemical analytical and microbiological methods. Therefore, this validated immunochromatographic test is expected to be suitable for routine analysis to ensure milk safety.

本研究采用Charm QUAD2®试剂盒开展免疫层析试验（immunochromatographic test），对生鲜牛乳中残留的大环内酯类（macrolides）与林可酰胺类（lincosamides）抗生素进行筛查。该试验的验证参数（选择性/特异性、检测能力（CCβ）以及耐用性）均符合[EC] 2021号法规的要求。本免疫层析试验的选择性通过微生物检测的阴性结果得以验证，其假阳性率为0%。针对牛乳中各类抗生素，该免疫层析试验的CCβ值如下：红霉素0.02 mg/kg、螺旋霉素0.1 mg/kg、替米考星0.025 mg/kg、泰乐菌素0.05 mg/kg、林可霉素0.15 mg/kg以及吡利霉素0.15 mg/kg。除林可霉素（与日本规定的最大残留限量（MRLs）持平）外，其余抗生素的测定CCβ值均低于各自对应的日本监管最大残留限量。除大环内酯类与林可酰胺类外的其他抗生素类物质不会干扰本试验的特异性，且批间重复性无显著差异。两名研究人员得到的检测结果亦无显著区别。最后，本试验应用于经泰乐菌素处理的奶牛采集的牛乳样本，检测结果呈阳性，与化学分析法和微生物检测法的结果一致。因此，本经过验证的免疫层析试验有望适用于保障牛乳安全的常规分析工作。