[Optimization and applicability evaluation of pretreatment method for rapid detection of Yersinia pestis in clinical samples by the up-converting phosphor technology-based immunochromatographic assay].

Objective

To optimize the pretreatment method of clinical samples in the up-converting phosphor technology-based immunochromatographic assay (UPT-ICA) for Yersinia pestis and evaluate its applicability.

Methods

A total of 464 clinical samples (including 215 serum samples, 60 cerebrospinal fluid samples, 42 pus samples, 88 sputum samples, and 59 tracheal secretion samples) were collected from the Sixth Medical Center of the PLA General Hospital and 35 samples of liver and spleen from natural foci of Yersinia pestis were provided by the Chinese Center for Disease Control and Prevention. After pretreatment, the samples were mixed with UPT-ICA diluent in a volume ratio of 1∶9 and then loaded onto the test strip to evaluate the specificity of UPT-ICA. 141 UPT-ICA negative clinical samples were randomly selected to prepare simulated positive samples with the final concentration of 1×105 CFU/ml of Yersinia pestis national reference material. The impact of the sample matrix on the detection performance of UPT-ICA was evaluated. The detection performance of the optimized UPT-ICA was comprehensively evaluated by optimizing the clinical sample pretreatment method, such as dilution ratio, digestion conditions, and sampling methods.

Results

The detection sensitivity of UPT-ICA for the Yersinia pestis national reference material was 1×105 CFU/ml, which was superior to that of the colloidal gold immunochromatographic assay. The specificity of UPT-ICA for tracheal secretion and cerebrospinal fluid samples was 100% (119/119). The false positive rates of clarified, hemolyzed, and lipemic serum were 2.35% (4/170), 8.70% (2/23), and 13.64% (3/22), respectively, and decreased to 0.59% (1/170) after 100-fold dilution. The false positive rate of the mixture of sputum and digestive fluid in a ratio of 1∶1 was 1.20% (1/83), and all turned negative after adjusting to 1∶2. Among the 42 pus samples, 3 false positives were detected. No false positives were found after all samples were collected using cotton swabs. The concentrations of liver and spleen homogenates at 10 mg/ml and 100 mg/ml had no effect on specificity. After optimizing the sample pretreatment method, the overall false positive rate of UPT-ICA for various clinical samples decreased from 7.14% (3/42) to 0.59% (1/170), and the detection rate of simulated positive samples reached 100% (171/171).

Conclusion

UPT-ICA can rapidly and accurately detect Yersinia pestis in clinical samples after optimizing the pretreatment method.