实验技术与管理

该研究旨在建立和优化一种针对实验动物肺炎克雷伯杆菌(Klebsiella pneumoniae,KP)的TaqMan实时荧光定量PCR(quantitative polymerase chain reaction,qPCR)检测方法，以提高检测的灵敏度、特异性和准确性。通过文献调研和序列比对，选取了KP的phoE基因作为目标序列，设计并合成一对特异性引物和探针，对qPCR反应体系和退火温度进行了系统优化，采用梯度稀释法测定了该方法的线性范围、灵敏度和检测限。通过分析不同浓度标准品的qPCR曲线，评估了该方法的特异性和方法再现性。最后将该方法用于各种临床样品检测。优化后的qPCR方法显示出良好的线性关系，线性范围为2.7×104～8.64 copies/μL，线性回归方程为y=–3.83x+42.865，相关系数(R²)为0.992。检测限达到8.64 copies/μL，表明该方法具有较高的灵敏度。特异性实验结果表明，该方法仅对KP产生特异性扩增，与其他常见病原体无交叉反应。统计不同操作人员对检测极限样品的10个平行Ct值，结果显示，Ct值的相对标准偏差均低于2%，说明方法具有良好的再现性。共检测26份临床样本，有3份临床样本为阳性，其他均为阴性。本研究成功建立了一种高灵敏度、高特异性的KP qPCR检测方法，为实验动物中的KP感染提供了一种快速、准确的分子诊断工具。

[Objective] Klebsiella pneumoniae(KP) is a gram-negative bacterium of the Enterobacteriaceae family, which is widely present in nature and one of the normal flora of the human and animal gastrointestinal and respiratory tracts. In hosts with compromised immune systems or chronic diseases, KP can cause various infections, including pneumonia, sepsis, and meningitis, posing a serious threat to human health. In addition, KP is one of the pathogens that must be detected and excluded in laboratory animals, particularly specific-pathogen-free mice. Therefore, to effectively control and prevent KP infections, rapid and accurate detection methods are particularly important. This study aims to establish a TaqMan real-time fluorescence quantitative PCR(qPCR) detection method for KP in laboratory animals and optimize the reaction system and program of this method to enhance its sensitivity, specificity, and accuracy. [Methods] Through literature research and sequence alignment, the phoE gene of KP is selected as the target sequence, and a pair of specific primers and probes are designed and synthesized using the software Oligo7. To achieve the lowest cycle number with the strongest fluorescence intensity, the reaction conditions are optimized by changing the final concentrations of primers and probes and the annealing temperature using KP-positive nucleic acid as a template. The linear range, sensitivity, and detection limit of the method are determined using a gradient dilution method. The specificity and method reproducibility are assessed by analyzing the qPCR curves of standards at different concentrations. Finally, the method is applied for detecting various clinical samples. [Results] The optimal annealing temperature for the optimized qPCR method was 50 ℃, with the optimal primer and probe concentrations at 0.2 μmol/L and 0.1 μmol/L, respectively. The optimized qPCR method demonstrated a good linear relationship, with a linear range of 2.7×104 to 8.64 copies/μL, a linear regression equation of y=-3.83x+42.865, and a correlation coefficient(R2) of 0.992. The detection limit of the method reached 8.64 copies/μL, indicating high sensitivity. The specificity test results showed that the method amplified KP specifically, with no cross-reaction with other common pathogens, indicating good specificity. Two different operators, at two different times, using different models of qPCR instruments, conducted 10 replicate experiments on samples at the detection limit. The statistical results of the 10 parallel experiments showed that the relative standard deviation of Ct values was below 2%, indicating good method reproducibility. A total of 26 clinical samples were tested, with 3 positive and 23 negative. [Conclusions] This study successfully established a highly sensitive and specific KP qPCR detection method, providing a rapid and accurate molecular diagnostic tool for KP infections in laboratory animals. This method helps to quickly identify KP at the early stages of infection, allowing for timely treatment measures and reducing the risk of disease transmission. Furthermore, this qPCR detection method can be further integrated into the routine health monitoring of laboratory animals, enhancing the biosafety level of laboratory animal facilities. At the same time, this method can also be extended to the detection of clinical samples, providing clinicians with a more accurate diagnostic basis and guiding clinical treatment and infection control.