Establishment and evaluation of a recombinase aided amplification-clustered regularly interspaced short palindromic repeats/associated protein system for detecting Klebsiella pneumoniae

doi:10.3877/cma.j.issn.1674-1358.2025.02.002

Abstract

Abstract:

Objective

To establish a method for detection of Klebsiella pneumoniae （KP） based on recombinase aided amplification （RAA）-clustered regularly interspaced short palindromic repeats （CRISPR）/CRISPR-associated protein （CRISPR-Cas13a） system.

Methods

CrRNA and RAA primer pairs were designed and screened for the detection of KP gene， while a rapid and accurate method for the detection of KP gene was developed based on RAA-CRISPR-Cas13a technology. The RAA-CRISPR and real time fluorescence quantitative PCR （RT-qPCR） methods were compared by the synthesized KP plasmid. Genomic DNA was extracted from clinical strains of Pseudomonas aeruginosa， Acinetobacter baumannii， Escherichia coli and Streptococcus pneumoniae for RAA-CRISPR detection to evaluate the specificity of this method.Total of 50 clinical samples （including 30 KP culture-positive samples and 20 KP-negative samples） collected from Peking Union Medical College Hospital， Chinese Academy of Medical Sciences， from April 2023 to March 2024 were detected for KP through both RAA-CRISPR and RT-qPCR. The sensitivity， positive agreement rate and negative agreement rate of both methods were compared， respectively. Statistical analysis of data was performed by ANOVA and paired t-tests.

Results

The optimal crRNA and RAA primers for KP detection were selected by KP-positive plasmids， and CRISPR-Cas13a-based KP detection method was established successfully. The detection sensitivity of CRISPR-Cas13a for KP plasmids reached 1 copy/μl， higher than that of RT-qPCR （10 copies/μl）. The specific evaluation results showed that there was no cross-reactivity with nontarget strains， indicating that CRISPR-Cas13a system was specific. Among the sample detection， the sensitivity of RAA-CRISPR and RT-qPCR were 100% （30/30） and 83.3% （25/30）， respectively， using bacterial culture and mass spectrometry technology as the gold standard. The positive concordance rate of both detection were 100% （30/30） and 83.3% （25/30）， and the negative concordance rate was 100%.

Conclusions

A method was established to accurately detect KP gene by RAA amplification technology and CRISPR-Cas13a system. This method can accurately detect KP gene， which can help diagnose KP infection for timely and effective treatment.

Key words: Klebsiella pneumoniae, Recombinase aid amplification, Clustered regularly interspaced short palindromic repeats sequences/relevant nucleases, Molecular diagnostic

Yaling Cao, Zhongjun Fang, Ling Xu, Binbin Jiang, Xiangying Zhang, Jing Huang, Feng Ren. Establishment and evaluation of a recombinase aided amplification-clustered regularly interspaced short palindromic repeats/associated protein system for detecting Klebsiella pneumoniae[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2025, 19(02): 70-76.

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Figures/Tables 6

References 28

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KP质粒稀释浓度	CRISPR-KP DNA（RFU）	t值	P值	RT-qPCR（CT）	t值	P值
10⁴拷贝/μl	2 255 541.13 ± 175 336.8	19.40	＜ 0.001	24.62 ± 0.64	33.28	＜ 0.001
10³拷贝/μl	2 377 344.21 ± 206 924.54	17.46	＜ 0.001	28.88 ± 0.35	31.73	＜ 0.001
10²拷贝/μl	2 280 285 ± 225 263.93	15.29	＜ 0.001	32.89 ± 0.64	16.48	＜ 0.001
10拷贝/μl	2 329 253.08 ± 100 727.42	34.97	＜ 0.001	39.20 ± 0.36	4.72	0.0092
1拷贝/μl	1 603 755.96 ± 124 504.98	18.24	＜ 0.001	40.06 ± 0.38	2.44	0.0713
阴性对照	290 058.33 ± 7 611.01	—	—	41.01 ± 0.56	—	—

KP质粒稀释浓度	CRISPR-KP DNA（RFU）	t值	P值	RT-qPCR（CT）	t值	P值
10⁴拷贝/μl	2 255 541.13 ± 175 336.8	19.40	＜ 0.001	24.62 ± 0.64	33.28	＜ 0.001
10³拷贝/μl	2 377 344.21 ± 206 924.54	17.46	＜ 0.001	28.88 ± 0.35	31.73	＜ 0.001
10²拷贝/μl	2 280 285 ± 225 263.93	15.29	＜ 0.001	32.89 ± 0.64	16.48	＜ 0.001
10拷贝/μl	2 329 253.08 ± 100 727.42	34.97	＜ 0.001	39.20 ± 0.36	4.72	0.0092
1拷贝/μl	1 603 755.96 ± 124 504.98	18.24	＜ 0.001	40.06 ± 0.38	2.44	0.0713
阴性对照	290 058.33 ± 7 611.01	—	—	41.01 ± 0.56	—	—

检测方法	细菌培养（株）		灵敏度（95%CI）	特异性（95%CI）	阳性符合率（95%CI）
检测方法	阳性	阴性	灵敏度（95%CI）	特异性（95%CI）	阳性符合率（95%CI）
CRISPR-Cas13a			100%（0.859～1.000）	100%（0.800～1.000）	100%（0.859～1.000）
阳性	30	0
阴性	0	20
RT-qPCR			83.3%（0.645～0.937）	100%（0.800～1.000）	83.3%（0.645～0.937）
阳性	25	0
阴性	5	20

检测方法	细菌培养（株）		灵敏度（95%CI）	特异性（95%CI）	阳性符合率（95%CI）
检测方法	阳性	阴性	灵敏度（95%CI）	特异性（95%CI）	阳性符合率（95%CI）
CRISPR-Cas13a			100%（0.859～1.000）	100%（0.800～1.000）	100%（0.859～1.000）
阳性	30	0
阴性	0	20
RT-qPCR			83.3%（0.645～0.937）	100%（0.800～1.000）	83.3%（0.645～0.937）
阳性	25	0
阴性	5	20

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