目的:建立Taq M an-MGB探针Real-tim e荧光PCR快速检测单增李斯特菌技术。方法:在对单增李斯特菌invA序列进行分析、比较基础上,设计一对特异性Taq M an-MGB探针法引物及探针,通过Real-tim ePCR反应条件和反应体系的优化,实现对单增李...目的:建立Taq M an-MGB探针Real-tim e荧光PCR快速检测单增李斯特菌技术。方法:在对单增李斯特菌invA序列进行分析、比较基础上,设计一对特异性Taq M an-MGB探针法引物及探针,通过Real-tim ePCR反应条件和反应体系的优化,实现对单增李斯特菌的快速检测;用克隆到pMD18-T载体上的李斯特菌invA基因阳参片段及不同菌株、食品标本验证方法的特异性和敏感性。结果:方法的灵敏性高,其循环阈值与模板浓度的对数值具有很好的对应关系,最低可检测57个拷贝数,经18 h增菌,可检测低至4.5 cfu/m l的细菌;特异性强,检测6株单增李斯特菌标准株和100株单增李斯特菌样品分离株的PCR循环域值(CT值)均小于25,而90株威氏李斯特菌、英诺克李斯特菌、绵羊李斯特菌以及非李斯特菌PCR循环域值(CT值)大于35;快速,最快1 h可出结果,实际样品20 h可出结果,而常规细菌培养法需要一周以上;对103份速冻米面食品进行检测,13份阳性,与常规方法无显著性差异(P>0.05)。结论:Taq M an-MGB探针的单增李斯特菌Real-tim e荧光PCR检测方法具有特异性强,敏感性高,易操作等优点,可推广应用。展开更多
Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resi...Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resistance necessitates updating diagnostic techniques to ensure higher sensitivity and specificity, especially with advancements in science and medicine. This study aimed to evaluate the prevalence of UTIs and antibiotic resistance profiles through urine culture, as well as to identify Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. in urine samples using a molecular approach with multiplex real-time PCR. From May 3 to July 25, 2023, at the Pietro Annigoni Biomolecular Research Center (CERBA) and Saint Camille Hospital of Ouagadougou (HOSCO), 209 urine samples collected from patients with suspected UTIs were analyzed using both urine culture and multiplex real-time PCR. Among the 209 patients, 52.15% were male and 47.85% female, with an average age of 46.87 ± 21.33 years. Urine cultures revealed an overall UTI prevalence of 23.44%, with a prevalence of 8.13% in men versus 15.31% in women (P = 0.023). The bacterial prevalence rates were as follows: Escherichia coli (12.92%), Klebsiella spp. (7.18%), Enterobacter cloacae (1.44%), Staphylococcus aureus (0.96%), and other bacteria. Klebsiella spp. demonstrated 100% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, while Escherichia coli showed 96.2% and 65.4% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, respectively. PCR analysis of the target bacteria revealed mono-infection prevalence rates of Klebsiella pneumoniae (10.39%), Klebsiella oxytoca (7.79%), and Acinetobacter spp. (7.79%), along with a co-infection prevalence rate of Klebsiella pneumoniae/Acinetobacter spp. (1.30%). This study demonstrated that PCR, with its high sensitivity and specificity, could effectively distinguish Klebsiella pneumoniae from Klebsiella oxytoca and detect Acinetobacter spp. in less than 24 hours—something urine culture alone could not achieve. The relative ease of automating urine PCR testing, combined with its diagnostic accuracy and rapid turnaround time, makes it a valuable addition to modern medical practice for the laboratory diagnosis of UTIs.展开更多
文摘目的:建立Taq M an-MGB探针Real-tim e荧光PCR快速检测单增李斯特菌技术。方法:在对单增李斯特菌invA序列进行分析、比较基础上,设计一对特异性Taq M an-MGB探针法引物及探针,通过Real-tim ePCR反应条件和反应体系的优化,实现对单增李斯特菌的快速检测;用克隆到pMD18-T载体上的李斯特菌invA基因阳参片段及不同菌株、食品标本验证方法的特异性和敏感性。结果:方法的灵敏性高,其循环阈值与模板浓度的对数值具有很好的对应关系,最低可检测57个拷贝数,经18 h增菌,可检测低至4.5 cfu/m l的细菌;特异性强,检测6株单增李斯特菌标准株和100株单增李斯特菌样品分离株的PCR循环域值(CT值)均小于25,而90株威氏李斯特菌、英诺克李斯特菌、绵羊李斯特菌以及非李斯特菌PCR循环域值(CT值)大于35;快速,最快1 h可出结果,实际样品20 h可出结果,而常规细菌培养法需要一周以上;对103份速冻米面食品进行检测,13份阳性,与常规方法无显著性差异(P>0.05)。结论:Taq M an-MGB探针的单增李斯特菌Real-tim e荧光PCR检测方法具有特异性强,敏感性高,易操作等优点,可推广应用。
文摘Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resistance necessitates updating diagnostic techniques to ensure higher sensitivity and specificity, especially with advancements in science and medicine. This study aimed to evaluate the prevalence of UTIs and antibiotic resistance profiles through urine culture, as well as to identify Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. in urine samples using a molecular approach with multiplex real-time PCR. From May 3 to July 25, 2023, at the Pietro Annigoni Biomolecular Research Center (CERBA) and Saint Camille Hospital of Ouagadougou (HOSCO), 209 urine samples collected from patients with suspected UTIs were analyzed using both urine culture and multiplex real-time PCR. Among the 209 patients, 52.15% were male and 47.85% female, with an average age of 46.87 ± 21.33 years. Urine cultures revealed an overall UTI prevalence of 23.44%, with a prevalence of 8.13% in men versus 15.31% in women (P = 0.023). The bacterial prevalence rates were as follows: Escherichia coli (12.92%), Klebsiella spp. (7.18%), Enterobacter cloacae (1.44%), Staphylococcus aureus (0.96%), and other bacteria. Klebsiella spp. demonstrated 100% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, while Escherichia coli showed 96.2% and 65.4% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, respectively. PCR analysis of the target bacteria revealed mono-infection prevalence rates of Klebsiella pneumoniae (10.39%), Klebsiella oxytoca (7.79%), and Acinetobacter spp. (7.79%), along with a co-infection prevalence rate of Klebsiella pneumoniae/Acinetobacter spp. (1.30%). This study demonstrated that PCR, with its high sensitivity and specificity, could effectively distinguish Klebsiella pneumoniae from Klebsiella oxytoca and detect Acinetobacter spp. in less than 24 hours—something urine culture alone could not achieve. The relative ease of automating urine PCR testing, combined with its diagnostic accuracy and rapid turnaround time, makes it a valuable addition to modern medical practice for the laboratory diagnosis of UTIs.
