基于纳米孔Cas9靶向测序的金黄色葡萄球菌快速检测与分型

doi:10.3969/j.issn.1674-8115.2025.06.013

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (6): 774-783.doi: 10.3969/j.issn.1674-8115.2025.06.013

• 论著 · 技术与方法 • 上一篇下一篇

基于纳米孔Cas9靶向测序的金黄色葡萄球菌快速检测与分型

潘抒凡¹^,², 杨栋磊¹^,²(), 王鹏飞¹^,²()

^1.上海交通大学医学院附属仁济医院，上海 200127
^2.上海交通大学医学院分子医学研究院，上海 200127

收稿日期:2024-10-24 接受日期:2025-02-28 出版日期:2025-06-28 发布日期:2025-06-28
通讯作者: 杨栋磊，副研究员，博士；电子信箱：dongleiyang@shsmu.edu.cn
王鹏飞，研究员，博士；电子信箱：pengfei.wang@sjtu.edu.cn。
作者简介:潘抒凡（2001—），女，博士生；电子信箱：shufan_alicepan@163.com。
基金资助:
上海地方高水平大学创新团队项目(SHSMU-ZLCX20212602)

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

PAN Shufan¹^,², YANG Donglei¹^,²(), WANG Pengfei¹^,²()

^1.Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
^2.Institute of Molecular Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

Received:2024-10-24 Accepted:2025-02-28 Online:2025-06-28 Published:2025-06-28
Contact: YANG Donglei, E-mail: dongleiyang@shsmu.edu.cn
WANG Pengfei, E-mail: pengfei.wang@sjtu.edu.cn.
Supported by:
Innovative Research Team of High-Level Local Universities in Shanghai(SHSMU-ZLCX20212602)

摘要/Abstract

摘要：

目的·利用纳米孔Cas9靶向测序（nanopore Cas9-targeted sequencing，nCATS）技术实现金黄色葡萄球菌的快速检测，并同时进行葡萄球菌蛋白A（staphylococcal protein A，spa）分型和葡萄球菌盒式染色体mec（staphylococcal cassette chromosome mec，SCCmec）分型。方法·以spa基因和SCCmec基因元件作为靶向测序的2个目标区域（region of interest，ROI），设计4种CRISPR RNA（crRNA），使其构成的Cas9核糖核蛋白（ribonucleoprotein，RNP）能够靶向切割2个ROI两侧的序列；针对每种crRNA设计42 bp的模拟靶标DNA。通过Cas9 RNP切割效率测试，筛选合适的crRNA，优化切割体系，选择切割反应时间和Cas9 RNP合成温度。提取耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus，MRSA）菌株基因组DNA，末端去磷酸化，在切割后的ROI两侧末端加上dA尾，与测序接头连接后利用纳米孔机器进行测序。对测序结果数据的质量值进行分析，并将测得的核酸序列分别与mecA、spa和SCCmec数据库进行比对，根据比对结果判断样本中是否含有金黄色葡萄球菌，是否为MRSA，并同时获得其spa分型和SCCmec分型。结果·共设计出2组crRNA，经电泳图灰度分析，筛选出切割效率更高的一组用于后续实验。切割效率优化结果显示，Cas9 RNP与靶标DNA比例为1∶1，切割反应时间为15 min，Cas9 RNP合成温度为25 ℃时，Cas9 RNP切割效率可达87.41%。nCATS测序质量值集中在15（Q15）和20（Q20）之间，表明测序准确度约为99%。测序结果与mecA、spa和SCCmec数据库比对后得出菌株样本的spa分型为t2，并且是SCCmec Ⅱ型。相同菌株样本经PCR扩增后测序和多重PCR验证，结果均与nCATS结果一致。结论·利用nCATS技术能够实现金黄色葡萄球菌的快速检测，并能同时获得spa分型和SCCmec分型。

关键词: 金黄色葡萄球菌, 纳米孔Cas9靶向测序, 细菌分型

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

中图分类号:

R446.5

潘抒凡, 杨栋磊, 王鹏飞. 基于纳米孔Cas9靶向测序的金黄色葡萄球菌快速检测与分型[J]. 上海交通大学学报（医学版）, 2025, 45(6): 774-783.

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, 45(6): 774-783.

图/表 11

表1 模拟靶标双链DNA序列

Tab 1 Simulated double-stranded target DNA sequences

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

图1 nCATS技术流程图Note： A. Schematic of the sample preparation process. B. Schematic of the nCATS mechanism. C. Schematic of the two ROIs and four crRNA cleavage sites. D. Sequencing data analysis workflow.

Fig 1 Schematic diagram of nCATS workflow

图2 根据切割效率筛选crRNANote： A. Cleavage products of eight crRNAs from two groups. B. Cleavage efficiencies of eight crRNAs from two groups. C. Comparison of cleavage efficiencies between the two groups of crRNAs. dsDNA—double-stranded DNA; ssDNA—single-stranded DNA.

Fig 2 Screening of crRNAs based on cleavage efficiency

图3 Cas9 RNP切割体系优化Note： A. Verification of cleavage products using the Cas9 RNP cleavage system (Cas9 RNP:dsDNA=1:2) at 20 ℃ for 60 min. B. Cleavage products verification of Cas9 RNP cleavage system (Cas9 RNP:dsDNA=1:1) with different cleavage time at 20 ℃. C. Cleavage efficiencies of Cas9 RNP under different ratios and different cleavage time.

Fig 3 Optimization of Cas9 RNP cleavage system

图4 Cas9 RNP合成温度筛选Note： A. Cleavage products of Cas9 RNP synthesized at 25 ℃, with different cleavage incubation times. B. Cleavage efficiencies of Cas9 RNP synthesized at 20 ℃ or 25 ℃, under different cleavage incubation times.

Fig 4 Screening of Cas9 RNP synthesis temperatures

表2 SCCmec 分型比对数据库所含序列GenBank登录号

Tab 2 GenBank accession numbers of sequences in SCCmec typing reference database

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

表3 PCR法鉴定 spa 分型使用的引物序列

Tab 3 Primer sequences used for spa typing by PCR

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

表4 多重PCR法鉴定SCCmec 分型使用的引物序列

Tab 4 Primer sequences used for SCCmec typing by multiplex PCR

mPCR	Primer	Sequence (5'→3')
mPCR1	mecA1	TGCTATCCACCCTCAAACAGG
	mecA2	AACGTTGTAACCACCCCAAGA
	ccrA1	AACCTATATCATCAATCAGTACGT
	ccrB123	ATTGCCTTGATAATAGCCTTCT
	ccrA2	TAAAGGCATCAATGCACAAACACT
	ccrA3	AGCTCAAAAGCAAGCAATAGAAT
	ccrA4	GTATCAATGCACCAGAACTT
	ccrB4	TTGCGACTCTCTTGGCGTTT
mPCR2	mecI	CATAACTTCCCATTCTGCAGATG
	mecA	ATATACCAAACCCGACAACTACA
	IS1272	ATGCTTAATGATAGCATCCGAATG
	IS431	TGAGGTTATTCAGATATTTCGATGT

表5 Cas9 RNP中的RNA序列

Tab 5 RNA sequences in Cas9 RNP

Name	Sequence (5'→3')
tracrRNA	AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU
spa-fw1-crRNA	AAUACAGUUGUACCGAUGAAGUUUUAGAGCUAUGCU
spa-rev1-crRNA	UCAAUUCGUAAACUAGGUGUGUUUUAGAGCUAUGCU
SCC2-fw1-crRNA	AUCGUAUUAAUAGAAUACAAGUUUUAGAGCUAUGCU
SCC2-rev1-crRNA	GGAAAGAUGCUAUCUUCCGAGUUUUAGAGCUAUGCU
spa-fw2-crRNA	AUGUCAUUUACUGUAUCACCGUUUUAGAGCUAUGCU
spa-rev2-crRNA	GGUACAUUACUUAUAUCUGGGUUUUAGAGCUAUGCU
SCC2-fw2-crRNA	AGAAUGUGUAAAAGUAGCUGGUUUUAGAGCUAUGCU
SCC2-rev2-crRNA	UGAACCAACGCAUGACCCAAGUUUUAGAGCUAUGCU

图5 nCATS测序数据质控结果Note： A. Histogram of sequencing read length. B. Plot of sequencing read length and average read quality.

Fig 5 Quality control results of nCATS sequencing data

图6 PCR和多重PCR法分型结果Note： A. Verification of spa amplicons. B. SCCmec typing results from multiplex PCR. RPT—repeat.

Fig 6 Typing results obtained using PCR and multiplex PCR methods

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基于纳米孔Cas9靶向测序的金黄色葡萄球菌快速检测与分型

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

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