机构：[a]Department of Clinical Laboratory , Shenzhen Shajing Hospital affiliated to Guangzhou Medical University , Shenzhen , China.深圳市康宁医院深圳医学信息中心[b]Dongguan Research Center, Traditional Chinese Medicine and New Drug Research Institute, Guangdong Medical University , Dongguan , China.[c]Department of Clinical Laboratory , The Second People's Hospital of Futian District , Shenzhen , China.深圳市康宁医院深圳医学信息中心
Background: Klebsiella pneumoniae is an opportunistic pathogen, causing an urgent threat to public
health because of resistance to the carbapenems. A rapid and accurate molecular method for the
detection of K. pneumoniae is required for guiding therapeutic treatment. Therefore, we aimed to
develop a multiplex real-time PCR assay for sensitive detection of K. pneumoniae.
Methods: Primers were designed according to the sequences of rcsA and 23S rRNA. Multiplex real-time
PCR assay was developed after primers screening and optimization of reaction conditions. The method
was clinically validated in 2754 sputum specimens tested by culture-based method were retested by the
PCR assay.
Results: Detection limit of the PCR assay was 2 cfu/reaction. Its sensitivity was 98.9% (94/95) on a panel
of 95 K. pneumoniae isolates, and its specificity was 97.9% (93/95) on a panel of 95 negative control
isolates. From the 2754 sputum specimens including 355 K. pneumoniae culture-positive specimens and
2399 K. pneumoniae culture-negative specimens, 349 of the 355 positive specimens were K.
pneumoniae positive, and 104 of 2399 negative specimens were K. pneumoniae positive with the PCR
assay. Of the 104 positive specimens, 75 specimens were confirmed by gene sequencing. The overall
results demonstrated that the diagnostic sensitivity and diagnostic specificity of our assay were 98.6%
(424/430) and 98.8% (2295/2324), respectively.
Conclusion: We report a high sensitive and specific multiplex PCR assay for rapid and accurate diagnosis
of K. pneumoniae.
基金：
This work was funded by grants from the Shenzhen Science and Technique Project (No.JCYJ20150402095058885) and the Shenzhen Baoan Science and Technique Project (No.
2015168)
第一作者机构：[a]Department of Clinical Laboratory , Shenzhen Shajing Hospital affiliated to Guangzhou Medical University , Shenzhen , China.
通讯作者：
推荐引用方式(GB/T 7714)：
Y Dou,L Li,J Du,et al.Development of a multiplex two-gene real-time PCR assay for accurate detection of Klebsiella pneumoniae.[J].BRITISH JOURNAL OF BIOMEDICAL SCIENCE.2019,76(1):42-45.doi:10.1080/09674845.2018.1499167.
APA：
Y Dou,L Li,J Du,Y He,R Chen...&H Liu.(2019).Development of a multiplex two-gene real-time PCR assay for accurate detection of Klebsiella pneumoniae..BRITISH JOURNAL OF BIOMEDICAL SCIENCE,76,(1)
MLA：
Y Dou,et al."Development of a multiplex two-gene real-time PCR assay for accurate detection of Klebsiella pneumoniae.".BRITISH JOURNAL OF BIOMEDICAL SCIENCE 76..1(2019):42-45
