Rapid detection and typing of Staphylococcus aureus based on nanopore Cas9-targeted sequencing

doi:10.3969/j.issn.1674-8115.2025.06.013

Abstract

Abstract:

Objective ·To achieve the rapid detection of Staphylococcus aureus using nanopore Cas9-targeted sequencing (nCATS) technology, and simultaneously perform staphylococcal protein A (spa) typing and staphylococcal cassette chromosome mec (SCCmec) typing. Methods ·The spa gene and SCCmec gene elements were selected as two regions of interest (ROIs) for targeted sequencing. Four types of CRISPR RNAs (crRNAs) were designed to form Cas9 ribonucleoproteins (RNPs) to cleave sequences flanking the two ROIs. For each crRNA, a 42 bp synthetic target DNA was designed. Appropriate crRNAs were screened, the cleavage system was optimized, and both the cleavage reaction time and Cas9 RNP synthesis temperature were determined based on the results of Cas9 RNP cleavage efficiency testing. Genomic DNA was extracted from a methicillin-resistant Staphylococcus aureus (MRSA) strain, and the ends flanking the cleaved ROIs were dephosphorylated and dA-tailed. Sequencing adapters were ligated, and sequencing was performed using a nanopore platform. The quality scores of the sequencing data were analyzed, and the obtained nucleic acid sequences were compared with those in the mecA, spa and SCCmec databases. Based on the comparison results, the presence of Staphylococcus aureus, MRSA or not, and spa and SCCmec types were determined. Results ·Two sets of crRNAs were designed. Based on grayscale analysis of electrophoresis results, the set with higher cleavage efficiency was selected for further experiments. Optimization showed that a 1∶1 ratio of Cas9 RNP to target DNA, a 15 min cleavage reaction time, and a Cas9 RNP synthesis temperature of 25 ℃ yielded a cleavage efficiency of 87.41%. nCATS sequencing quality scores ranged between 15 (Q15) and 20 (Q20), indicating an approximate sequencing accuracy of 99%. Sequence comparisons with the mecA, spa and SCCmec databases revealed that the strain's spa type was t2 and its SCCmec type was Ⅱ. These results were consistent with those obtained by PCR amplification sequencing and multiplex PCR. Conclusion ·The nCATS technique enables rapid detection of Staphylococcus aureus, while simultaneously providing spa and SCCmec typing information.

Key words: Staphylococcus aureus, nanopore Cas9-targeted sequencing (nCATS), bacterial typing

CLC Number:

R446.5

PAN Shufan, YANG Donglei, WANG Pengfei. Rapid detection and typing of Staphylococcus aureus based on nanopore Cas9-targeted sequencing[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025,(6): 774-783.

Figures/Tables 11

TrendMD

References 25

[1]	PALAVECINO E L. Clinical, epidemiologic, and laboratory aspects of methicillin-resistant Staphylococcus aureus infections[J]. Methods Mol Biol, 2020, 2069: 1-28.
[2]	郭燕, 胡付品, 朱德妹, 等. 2022年CHINET三级医院细菌耐药监测[J]. 中国感染与化疗杂志, 2024, 24(3): 277-286.
	GUO Y, HU F P, ZHU D M, et al. Surveillance of bacterial resistance in tertiary hospitals across China: results of CHINET Antimicrobial Resistance Surveillance Program in 2022[J]. Chinese Journal of Infection and Chemotherapy, 2024, 24(3): 277-286.
[3]	邱杰, 苏明宽, 吴海英. 葡萄球菌A蛋白快速检测金黄色葡萄球菌的临床监测与研究[J]. 临床合理用药杂志, 2018, 11(11): 100-101.
	QIU J, SU M K, WU H Y. Clinical monitoring and research on the rapid detection of Staphylococcus aureus by staphylococcal protein A[J]. Chinese Journal of Clinical Rational Drug Use, 2018, 11(11): 100-101.
[4]	RIGI G, GHAEDMOHAMMADI S, AHMADIAN G. A comprehensive review on staphylococcal protein A (SpA): its production and applications[J]. Biotechnol Appl Biochem, 2019, 66(3): 454-464.
[5]	邹自英, 韩黎, 熊杰, 等. 金黄色葡萄球菌临床分离株spa分型和耐药特征研究[J]. 中国感染与化疗杂志, 2014, 14(2): 142-145.
	ZOU Z Y, HAN L, XIONG J, et al. spa Typing and resistance profile of Staphylococcus aureus isolated from clinical specimens[J]. Chinese Journal of Infection and Chemotherapy, 2014, 14(2): 142-145.
[6]	HALLIN M, FRIEDRICH A W, STRUELENS M J. spa Typing for epidemiological surveillance of Staphylococcus aureus[J]. Methods Mol Biol, 2009, 551: 189-202.
[7]	GUO Y L, SONG G H, SUN M L, et al. Prevalence and therapies of antibiotic-resistance in Staphylococcus aureus[J]. Front Cell Infect Microbiol, 2020, 10: 107.
[8]	GIULIERI S G, TONG S Y C, WILLIAMSON D A. Using genomics to understand meticillin- and vancomycin-resistant Staphylococcus aureus infections[J]. Microb Genom, 2020, 6(1): e000324.
[9]	LAM J C, STOKES W. The golden grapes of wrath-Staphylococcus aureus bacteremia: a clinical review[J]. Am J Med, 2023, 136(1): 19-26.
[10]	LAKHUNDI S, ZHANG K Y. Methicillin-resistant Staphylococcus aureus: molecular characterization, evolution, and epidemiology[J]. Clin Microbiol Rev, 2018, 31(4): e00020-18.
[11]	武杰, 赵建平. 耐甲氧西林金黄色葡萄球菌分型方法研究进展[J]. 中国感染与化疗杂志, 2021, 21(2): 235-240.
	WU J, ZHAO J P. Advances in typing methods of methicillin-resistant Staphylococcus aureus[J]. Chinese Journal of Infection and Chemotherapy, 2021, 21(2): 235-240.
[12]	CHARALAMPOUS T, KAY G L, RICHARDSON H, et al. Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection[J]. Nat Biotechnol, 2019, 37(7): 783-792.
[13]	WANG M, FU A S, HU B, et al. Nanopore targeted sequencing for the accurate and comprehensive detection of SARS-CoV-2 and other respiratory viruses[J]. Small, 2020, 16(32): e2002169.
[14]	SLIZOVSKIY I B, OLIVA M, SETTLE J K, et al. Target-enriched long-read sequencing (TELSeq) contextualizes antimicrobial resistance genes in metagenomes[J]. Microbiome, 2022, 10(1): 185.
[15]	QUAN J, LANGELIER C, KUCHTA A, et al. FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences[J]. Nucleic Acids Res, 2019, 47(14): e83.
[16]	SERPA P H, DENG X D, ABDELGHANY M, et al. Metagenomic prediction of antimicrobial resistance in critically ill patients with lower respiratory tract infections[J]. Genome Med, 2022, 14(1): 74.
[17]	WANG S W, GAO C, ZHENG Y M, et al. Current applications and future perspective of CRISPR/Cas9 gene editing in cancer[J]. Mol Cancer, 2022, 21(1): 57.
[18]	WANG Y H, ZHAO Y, BOLLAS A, et al. Nanopore sequencing technology, bioinformatics and applications[J]. Nat Biotechnol, 2021, 39(11): 1348-1365.
[19]	杨影, 谢水莲, 万绍贵, 等. 基于Cas9靶向富集的纳米孔高通量基因测序技术的应用进展[J]. 赣南医学院学报, 2022, 42(4): 342-347.
	YANG Y, XIE S L, WAN S G, et al. The applications of high-throughput nanopore based Cas9-targeted sequencing [J]. Journal of Gannan Medical University, 2022, 42(4): 342-347.
[20]	GILPATRICK T, LEE I, GRAHAM J E, et al. Targeted nanopore sequencing with Cas9-guided adapter ligation[J]. Nat Biotechnol, 2020, 38(4): 433-438.
[21]	BARTELS M D, PETERSEN A, WORNING P, et al. Comparing whole-genome sequencing with Sanger sequencing for spa typing of methicillin-resistant Staphylococcus aureus[J]. J Clin Microbiol, 2014, 52(12): 4305-4308.
[22]	YAMAGUCHI T, ONO D, SATO A. Staphylococcal cassette chromosome mec (SCCmec) analysis of MRSA[J]. Methods Mol Biol, 2020, 2069: 59-78.
[23]	窦宇红, 梁鸿, 何玥, 等. qPCR快速检测金黄色葡萄球菌及MRSA方法的建立及评价[J]. 中国感染控制杂志, 2018, 17(9): 764-769.
	DOU Y H, LIANG H, HE Y, et al. Establishment and evaluation of a quantitative real-time PCR assay for rapid detection of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus[J]. Chinese Journal of Infection Control, 2018, 17(9): 764-769.
[24]	CHIBA M, AOYAGI T, YOSHIDA M, et al. Evaluation of the performance of GeneSoC^®, a novel rapid real-time PCR system, to detect Staphylococcus aureus and methicillin resistance in blood cultures[J]. J Infect Chemother, 2023, 29(7): 718-721.
[25]	BILGRAU A E, FALGREEN S, PETERSEN A, et al. Unaccounted uncertainty from qPCR efficiency estimates entails uncontrolled false positive rates[J]. BMC Bioinformatics, 2016, 17: 159.

crRNA	Strand	Corresponding simulated target DNA (5'→3')
spa-fw1-crRNA	+	ACCAAATACAGTTGTACCGATGAATGGATTTTCTTCACCAGT
	-	ACTGGTGAAGAAAATCCATTCATCGGTACAACTGTATTTGGT
spa-fw2-crRNA	+	TGCAATGTCATTTACTGTATCACCAGGTTTAACGACATGTAC
	-	GTACATGTCGTTAAACCTGGTGATACAGTAAATGACATTGCA
spa-rev1-crRNA	+	GTTACAGATGCAATACCTACACCTAGTTTACGAATTGAATAA
	-	TTATTCAATTCGTAAACTAGGTGTAGGTATTGCATCTGTAAC
spa-rev2-crRNA	+	GCAGCAGGTGTTACGCCACCAGATATAAGTAATGTACCTAAA
	-	TTTAGGTACATTACTTATATCTGGTGGCGTAACACCTGCTGC
SCC2-fw1-crRNA	+	ATTAATCGTATTAATAGAATACAAGGGCAACTAAATGGAATT
	-	AATTCCATTTAGTTGCCCTTGTATTCTATTAATACGATTAAT
SCC2-fw2-crRNA	+	TAATAGAATGTGTAAAAGTAGCTGCGGATGATGAAGAAAGCT
	-	AGCTTTCTTCATCATCCGCAGCTACTTTTACACATTCTATTA
SCC2-rev1-crRNA	+	AATTCTTGGGCCAATCCTTCGGAAGATAGCATCTTTCCTTGT
	-	ACAAGGAAAGATGCTATCTTCCGAAGGATTGGCCCAAGAATT
SCC2-rev2-crRNA	+	TACAAAGTCGCTTTGCCCTTGGGTCATGCGTTGGTTCAATTC
	-	GAATTGAACCAACGCATGACCCAAGGGCAAAGCGACTTTGTA

crRNA	Strand	Corresponding simulated target DNA (5'→3')
spa-fw1-crRNA	+	ACCAAATACAGTTGTACCGATGAATGGATTTTCTTCACCAGT
	-	ACTGGTGAAGAAAATCCATTCATCGGTACAACTGTATTTGGT
spa-fw2-crRNA	+	TGCAATGTCATTTACTGTATCACCAGGTTTAACGACATGTAC
	-	GTACATGTCGTTAAACCTGGTGATACAGTAAATGACATTGCA
spa-rev1-crRNA	+	GTTACAGATGCAATACCTACACCTAGTTTACGAATTGAATAA
	-	TTATTCAATTCGTAAACTAGGTGTAGGTATTGCATCTGTAAC
spa-rev2-crRNA	+	GCAGCAGGTGTTACGCCACCAGATATAAGTAATGTACCTAAA
	-	TTTAGGTACATTACTTATATCTGGTGGCGTAACACCTGCTGC
SCC2-fw1-crRNA	+	ATTAATCGTATTAATAGAATACAAGGGCAACTAAATGGAATT
	-	AATTCCATTTAGTTGCCCTTGTATTCTATTAATACGATTAAT
SCC2-fw2-crRNA	+	TAATAGAATGTGTAAAAGTAGCTGCGGATGATGAAGAAAGCT
	-	AGCTTTCTTCATCATCCGCAGCTACTTTTACACATTCTATTA
SCC2-rev1-crRNA	+	AATTCTTGGGCCAATCCTTCGGAAGATAGCATCTTTCCTTGT
	-	ACAAGGAAAGATGCTATCTTCCGAAGGATTGGCCCAAGAATT
SCC2-rev2-crRNA	+	TACAAAGTCGCTTTGCCCTTGGGTCATGCGTTGGTTCAATTC
	-	GAATTGAACCAACGCATGACCCAAGGGCAAAGCGACTTTGTA

SCCmec type	GenBank No.
SCCmec Ⅱ	AB127982.1
	AB261975.1
	AB433542.1
	AB435013.1
	AB435014.1
	AB665981.1
	AJ810120.1
	D86934.2
SCCmec Ⅲ	AB038513.1
	AB037671.1
	AB047088.2
	AB047089.2
	AB425427.1

SCCmec type	GenBank No.
SCCmec Ⅱ	AB127982.1
	AB261975.1
	AB433542.1
	AB435013.1
	AB435014.1
	AB665981.1
	AJ810120.1
	D86934.2
SCCmec Ⅲ	AB038513.1
	AB037671.1
	AB047088.2
	AB047089.2
	AB425427.1

Primer	Sequence (5'→3')
spa-1113f	TAAAGACGATCCTTCGGTGAGC
spa-1514r	CAGTAGTGCCGTTTGCTT