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Article(id=1204800732894572624, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1204800727341310425, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250354, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1745856000000, receivedDateStr=2025-04-29, revisedDate=null, revisedDateStr=null, acceptedDate=1749657600000, acceptedDateStr=2025-06-12, onlineDate=1765176478836, onlineDateStr=2025-12-08, pubDate=1764777600000, pubDateStr=2025-12-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1765176478836, onlineIssueDateStr=2025-12-08, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1765176478836, creator=13701087609, updateTime=1765176478836, updator=13701087609, issue=Issue{id=1204800727341310425, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='12', pageStart='5191', pageEnd='5649', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1765176477513, creator=13701087609, updateTime=1765176611928, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1204801291189986067, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1204800727341310425, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1204801291189986068, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1204800727341310425, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=5339, endPage=5351, ext={EN=ArticleExt(id=1204800733238505573, articleId=1204800732894572624, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Species-level identification of Burkholderia cepacia complex through hutC sequence analysis, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

Objective To explore the application of hutC sequence analysis in the identification of Burkholderia cepacia complex (Bcc) at the species level. Methods We compared the sequences of hutC and the amino acid sequences of the encoded proteins and conducted phylogenetic analysis to theoretically assess the feasibility of using hutC for species-level identification of Bcc. Primers targeting the hutC of Bcc were designed, and the amplification conditions were optimized. With those of standard strains as templates, the hutC sequences of representative Bcc species were amplified. The sequencing results were compared with NCBI sequences for phylogenetic analysis to validate the theoretical hypothesis. Additionally, single nucleotide polymorphism analysis of hutC sequences was performed to identify species-level characteristic barcodes of Bcc. Results The hutC gene was relatively conserved between Bcc and non-Bcc. With the designed primers and amplification conditions, a 692 bp fragment of hutC was successfully amplified from 12 Bcc standard strains. Except for one strain with taxonomic errors, the remaining 11 strains had the comparison results consistent with those from the culture collection centers. The phylogenetic analysis based on hutC showed that different Bcc species could be clustered with high bootstrap values. A 12 nucleotide characteristic barcode of hutC was identified, which can rapidly distinguish different species of Bcc. Conclusion The gene hutC can serve as a new housekeeping gene target for accurate identification of Bcc at the complex and species levels.

, correspAuthors=Shihong MA, authorNote=null, correspAuthorsNote=
*E-mail:
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目的 探讨利用hutC基因对洋葱伯克霍尔德菌群(Burkholderia cepacia complex, Bcc)进行种水平鉴定的可行性。 方法 对hutC基因序列及其编码蛋白的氨基酸序列进行比对和系统发育分析，从理论上评估用于Bcc种水平鉴定的可行性。针对Bcc的hutC基因设计该基因扩增引物，以标准菌株为模板扩增Bcc代表菌种的hutC基因，将测序结果与NCBI序列进行比对和系统发育分析以验证前述理论假设。此外，对hutC基因序列进行单核苷酸多态性分析，查找Bcc种水平的特征条码。 结果 hutC基因在Bcc和非Bcc菌种间相对保守。采用所设计的引物和扩增条件可扩增出12株Bcc标准菌株长度为692 bp的hutC基因序列。除1株分类学有误的菌株外，其余11株的比对结果均与保藏中心一致。对hutC基因的系统发育分析结果表明，不同Bcc菌种能够以较高的自展值聚类。由12个核苷酸组成的hutC基因特征条码可快速区分不同的Bcc菌种。 结论 hutC基因可作为Bcc鉴定的新型看家基因靶点，用于Bcc菌群和菌种水平的准确鉴定。

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orderNo=3, keyword=看家基因), Keyword(id=1217784599271358647, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=CN, orderNo=4, keyword=微生物鉴定)], refs=[Reference(id=1217784602589053335, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, doi=null, pmid=null, pmcid=null, year=1981, volume=31, issue=4, pageStart=479, pageEnd=481, url=null, language=null, rfNumber=[1], rfOrder=0, authorNames=PALLERONI NJ, HOLMES B, journalName=International Journal of Systematic Evolutionary Microbiology, refType=null, unstructuredReference=PALLERONI NJ, HOLMES B. 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Microbiology and Molecular Biology Reviews, 2012, 76(3): 565-584., articleTitle=Regulation of the histidine utilization (hut) system in bacteria, refAbstract=null), Reference(id=1217784606351344217, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, doi=null, pmid=null, pmcid=null, year=2014, volume=5, issue=null, pageStart=429, pageEnd=null, url=null, language=null, rfNumber=[28], rfOrder=30, authorNames=SAWANA A, ADEOLU M, GUPTA RS, journalName=Frontiers in Genetics, refType=null, unstructuredReference=SAWANA A, ADEOLU M, GUPTA RS. Molecular signatures and phylogenomic analysis of the genus Burkholderia: proposal for division of this genus into the emended genus Burkholderia containing pathogenic organisms and a new genus Paraburkholderia gen. nov. harboring environmental species[J]. Frontiers in Genetics, 2014, 5: 429., articleTitle=Molecular signatures and phylogenomic analysis of the genus Burkholderia: proposal for division of this genus into the emended genus Burkholderia containing pathogenic organisms and a new genus Paraburkholderia gen. nov. harboring environmental species, refAbstract=null)], funds=[Fund(id=1217784602165428610, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, awardId=2025HYZX04, language=EN, fundingSource=NIFDC Key Laboratory of Chemical Drug Analysis Project(2025HYZX04), fundOrder=null, country=null), Fund(id=1217784602257703304, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, awardId=2025HYZX04, language=CN, fundingSource=中国食品药品检定研究院化学药品检定所重点实验室项目(2025HYZX04), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1217784595983025074, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, xref=1., ext=[AuthorCompanyExt(id=1217784595999802291, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, companyId=1217784595983025074, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.National Institutes for Food and Drug Control, Beijing, China), AuthorCompanyExt(id=1217784596020773812, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, companyId=1217784595983025074, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.中国食品药品检定研究院，北京)]), AuthorCompany(id=1217784596129825730, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, xref=2., ext=[AuthorCompanyExt(id=1217784596138214339, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, companyId=1217784596129825730, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.China Pharmaceutical University, Nanjing, Jiangsu, China), AuthorCompanyExt(id=1217784596142408644, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, companyId=1217784596129825730, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.中国药科大学，江苏 南京)])], figs=[ArticleFig(id=1217784599560765650, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=EN, label=Figure 1, caption=Maximum likelihood phylogenetic tree of the hutC histidine utilization repressor amino acid sequences from Bcc and closely related species. Bootstrap values>50% are shown, and the accession number of each sequence is shown in parentheses., figureFileSmall=9WSWjXWczQctnq5XA5G/Pg==, figureFileBig=HvnNKdlq5dSQAG/m6ALGKQ==, tableContent=null), ArticleFig(id=1217784599682400473, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=CN, label=图1, caption=Bcc及其近缘菌种组氨酸利用阻遏物C氨基酸序列系统发育最大似然树。显示自展值大于50%，每条序列的登记号在括号中显示。, figureFileSmall=9WSWjXWczQctnq5XA5G/Pg==, figureFileBig=HvnNKdlq5dSQAG/m6ALGKQ==, tableContent=null), ArticleFig(id=1217784599871144170, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=EN, label=Figure 2, caption=The annealing temperature of PCR reactions was investigated. For primer pair P3, lanes 1-8 corresponded to annealing temperatures of 48.0, 48.5, 49.3, 50.7, 52.3, 53.6, 54.5 and 55.0 ℃, respectively. For primer pair P4, samples 9-16 corresponded to the same annealing temperatures as samples 1-8. M represents the DNA marker., figureFileSmall=VU9Vu0ff4hLoCbwyYSd1uQ==, figureFileBig=cMduF+NB4kRPTYpnCDvWXw==, tableContent=null), ArticleFig(id=1217784600013750513, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=CN, label=图2, caption=PCR反应退火温度考察。引物对P3对应泳道1-8，依次为退火温度48.0、48.5、49.3、50.7、52.3、53.6、54.5、55.0 ℃；引物对P4对应泳道9-16，顺序同泳道1-8；M：DNA marker。, figureFileSmall=VU9Vu0ff4hLoCbwyYSd1uQ==, figureFileBig=cMduF+NB4kRPTYpnCDvWXw==, tableContent=null), ArticleFig(id=1217784600160551162, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=EN, label=Figure 3, caption=PCR amplification and electrophoresis detection results of hutC gene in test strains. M represents the marker; Lanes 1-14 corresponded to ATCC 25416, CMCC(B) 23005, CMCC(B) 23006, ATCC BAA-246, ATCC BAA-245, CMCC(B) 23010, CGMCC 1.2982, CGMCC 1.2872, ATCC BAA-247, CGMCC 1.3816, CGMCC 1.10511, CICC 24958, Negative control, and CICC 23882, respectively., figureFileSmall=WKJAZKskCadzvF3BJx+/KQ==, figureFileBig=UTzYqbtRwyhX07kQz98++w==, tableContent=null), ArticleFig(id=1217784600257020159, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=CN, label=图3, caption=试验菌株 hutC 基因扩增电泳检测结果, figureFileSmall=WKJAZKskCadzvF3BJx+/KQ==, figureFileBig=UTzYqbtRwyhX07kQz98++w==, tableContent=null), ArticleFig(id=1217784600374460678, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=EN, label=Figure 4, caption=Phylogenetic tree of hutC sequences at different Bcc species levels. Bootstrap values above 50% are shown, and different colors represent different Bcc species-level taxa. Type strains are indicated in , and the tested strains are indicated in bold., figureFileSmall=zVKiQhFkKrwF31tMa38pSA==, figureFileBig=hnpEOn96K9SKyJJy8LXgjw==, tableContent=null), ArticleFig(id=1217784600483512593, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=CN, label=图4, caption=Bcc不同种水平菌株 hutC 序列系统发育树。显示自展值大于50%，不同颜色代表不同Bcc种水平分类单元，模式菌株以 标识，试验菌株以加粗字体显示。, figureFileSmall=zVKiQhFkKrwF31tMa38pSA==, figureFileBig=hnpEOn96K9SKyJJy8LXgjw==, tableContent=null), ArticleFig(id=1217784600588370204, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=EN, label=Table 1, caption=

Strains used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
Type strain numberName
ATCC 25416B. cepacia
ATCC BAA-245B. cenocepacia
ATCC BAA-246B. dolosa
ATCC BAA-247B. multivorans
CICC 23882B. contaminans
CMCC(B) 23005B. cepacia
CMCC(B) 23006B. cenocepacia
CMCC(B) 23010=CICC 24958B. aenigmatica
CGMCC 1.2872B. stabilis
CGMCC 1.2982B. vietnamiensis
CGMCC 1.10511B. ambifaria
CGMCC 1.3816B. pyrrocinia
), ArticleFig(id=1217784600676450597, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=CN, label=表1, caption=

本研究所用菌株

, figureFileSmall=null, figureFileBig=null, tableContent=
Type strain numberName
ATCC 25416B. cepacia
ATCC BAA-245B. cenocepacia
ATCC BAA-246B. dolosa
ATCC BAA-247B. multivorans
CICC 23882B. contaminans
CMCC(B) 23005B. cepacia
CMCC(B) 23006B. cenocepacia
CMCC(B) 23010=CICC 24958B. aenigmatica
CGMCC 1.2872B. stabilis
CGMCC 1.2982B. vietnamiensis
CGMCC 1.10511B. ambifaria
CGMCC 1.3816B. pyrrocinia
), ArticleFig(id=1217784600793891115, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=EN, label=Table 2, caption=

Primers used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
NumberPrimers namePrimer sequences (5′→3′)*
P1hutC-F-702GGCGCCCGGCTTGAA
hutC-R-702GCACCGTTCCAGCAGATCAA
P2hutC-FJ-702SGCGCCSGGCTTGAA
hutC-RJ-702GCRCCGTTCCAGCAGATCAA
P3hutC-F-692TTGAAGCGCGAGCCGAG
hutC-R-692GCACCGTTCCAGCAGATCAAG
P4hutC-FJ-692CTTGAAGCGCGAVCCGAG
hutC-RJ-692GCACCGTTCCAGCARATCAAG
), ArticleFig(id=1217784600886165812, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=CN, label=表2, caption=

本研究所用引物

, figureFileSmall=null, figureFileBig=null, tableContent=
NumberPrimers namePrimer sequences (5′→3′)*
P1hutC-F-702GGCGCCCGGCTTGAA
hutC-R-702GCACCGTTCCAGCAGATCAA
P2hutC-FJ-702SGCGCCSGGCTTGAA
hutC-RJ-702GCRCCGTTCCAGCAGATCAA
P3hutC-F-692TTGAAGCGCGAGCCGAG
hutC-R-692GCACCGTTCCAGCAGATCAAG
P4hutC-FJ-692CTTGAAGCGCGAVCCGAG
hutC-RJ-692GCACCGTTCCAGCARATCAAG
), ArticleFig(id=1217784601045549373, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=EN, label=Table 3, caption=

Identification results of test strains

, figureFileSmall=null, figureFileBig=null, tableContent=
NumberBiolog16S rRNA (similar values, %)recA (similar values, %)
ATCC 25416B. pyrrocinia/cepaciaB. cepacia (99.93)B. cepacia (99.79)
ATCC BAA-245B. cenocepaciaB. contaminans (99.71)B. cenocepacia (99.89)
ATCC BAA-246B. dolosaB. dolosa (99.86)B. dolosa (99.79)
ATCC BAA-247B. multivoransB. multivorans (100.00)B. multivorans (99.79)
CICC 23882B. contaminansB. lata (99.93)B. contaminans (99.70)
CMCC(B) 23005B. pyrrocinia/cepaciaB. territorii (99.79)B. cepacia (99.80)
CMCC(B) 23006B. dolosaB. cepacia (99.72)B. cenocepacia (100.00)
CMCC(B) 23010B. ambifaria/cepaciaB. arboris (99.79)B. lata (98.40)
CGMCC 1.2872B. stabilis/cepaciaB. pyrrocinia (99.93)B. stabilis (99.80)
CGMCC 1.2982B. vietnamiensisB. vietnamiensis (99.86)B. vietnamiensis (99.70)
CGMCC 1.10511Pseudomonas syringae pv. viburniB. ambifaria (99.86)B. ambifaria (98.91)
CGMCC 1.3816B. ambifaria/cepaciaB. ambifaria (99.93)B. ambifaria (100.00)
), ArticleFig(id=1217784601146212678, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=CN, label=表3, caption=

实验菌株生化及保守序列鉴定结果

, figureFileSmall=null, figureFileBig=null, tableContent=
NumberBiolog16S rRNA (similar values, %)recA (similar values, %)
ATCC 25416B. pyrrocinia/cepaciaB. cepacia (99.93)B. cepacia (99.79)
ATCC BAA-245B. cenocepaciaB. contaminans (99.71)B. cenocepacia (99.89)
ATCC BAA-246B. dolosaB. dolosa (99.86)B. dolosa (99.79)
ATCC BAA-247B. multivoransB. multivorans (100.00)B. multivorans (99.79)
CICC 23882B. contaminansB. lata (99.93)B. contaminans (99.70)
CMCC(B) 23005B. pyrrocinia/cepaciaB. territorii (99.79)B. cepacia (99.80)
CMCC(B) 23006B. dolosaB. cepacia (99.72)B. cenocepacia (100.00)
CMCC(B) 23010B. ambifaria/cepaciaB. arboris (99.79)B. lata (98.40)
CGMCC 1.2872B. stabilis/cepaciaB. pyrrocinia (99.93)B. stabilis (99.80)
CGMCC 1.2982B. vietnamiensisB. vietnamiensis (99.86)B. vietnamiensis (99.70)
CGMCC 1.10511Pseudomonas syringae pv. viburniB. ambifaria (99.86)B. ambifaria (98.91)
CGMCC 1.3816B. ambifaria/cepaciaB. ambifaria (99.93)B. ambifaria (100.00)
), ArticleFig(id=1217784601267847504, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=EN, label=Table 4, caption=

Sequences alignment results of hutC gene in test strains

, figureFileSmall=null, figureFileBig=null, tableContent=
Strain numberhutC (similar values, %)
ATCC 25416B. cepacia (100.00)
ATCC BAA-245B. cenocepacia (99.71)
ATCC BAA-246B. dolosa (100.00)
ATCC BAA-247B. multivorans (100.00)
CICC 23882B. contaminans (100.00)
CMCC(B) 23005B. cepacia (100.00)
CMCC(B) 23006B. cenocepacia (100.00)
CMCC(B) 23010B. aenigmatica (99.71)
CGMCC 1.2872B. stabilis (100.00)
CGMCC 1.2982B. vietnamiensis (100.00)
CGMCC 1.10511B. ambifaria (99.57)
CGMCC 1.3816B. ambifaria (100.00)
), ArticleFig(id=1217784601519505754, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=CN, label=表4, caption=

试验菌株 hutC 序列比对结果

, figureFileSmall=null, figureFileBig=null, tableContent=
Strain numberhutC (similar values, %)
ATCC 25416B. cepacia (100.00)
ATCC BAA-245B. cenocepacia (99.71)
ATCC BAA-246B. dolosa (100.00)
ATCC BAA-247B. multivorans (100.00)
CICC 23882B. contaminans (100.00)
CMCC(B) 23005B. cepacia (100.00)
CMCC(B) 23006B. cenocepacia (100.00)
CMCC(B) 23010B. aenigmatica (99.71)
CGMCC 1.2872B. stabilis (100.00)
CGMCC 1.2982B. vietnamiensis (100.00)
CGMCC 1.10511B. ambifaria (99.57)
CGMCC 1.3816B. ambifaria (100.00)
), ArticleFig(id=1217784601615974754, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=EN, label=Table 5, caption=

Characteristic base barcode in hutC gene sequences for identifying Bcc at the species level

, figureFileSmall=null, figureFileBig=null, tableContent=
Name of Bcc speciesSingle nucleotide polymorphism (SNP) sites (relative to the full-length hutC sequence)
456459478481482498506523546569579647
B. cepaciaCTCGTGATGGGT
B. cenocepaciaCCGGCGATCGGT
B. arborisCCCGCGTTTCGT
B. seminalisCCCACGATCTGT
B. pyrrociniaCTCACGATCGGT
B. stabilisMCCACGTTCGGT
B. metallicaCCCACGATGGGT
B. aenigmaticaCTCGTGATGGAT
B. lataCYCGTGATGGGA
B. contaminansCCCGTGTTGGGA
B. purquaeCCCGTAAGGGGT
B. paludisCTCGTGTGCGCT
B. pseudomultivoransGTCACATTCTGT
B. diffusaMACGCATTGGGT
B. territoriiCAGGTATTGGGT
B. ambifariaCACGCATTCGGT
B. anthinaGACACATTCGGT
B. latensGACACATTGGGT
B. dolosaCACGCGATCGGT
B. multivoransCMMCAGAGCCGT
B. vietnamiensisCTGGTATCCGAT
B. stagnalisCCGCAGATGAGT
B. ubonensisGCCRGGASCAGT
B. catarinensisCCCGCGAGGGGT
B. solaCTCACGACCGGT
B. semiaridaCCCACGAGCGGT
B. orbicolaCCCACGATCGGT
), ArticleFig(id=1217784601737609576, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=CN, label=表5, caption=

hutC 基因序列中用于Bcc种水平鉴定的特征碱基条码

, figureFileSmall=null, figureFileBig=null, tableContent=
Name of Bcc speciesSingle nucleotide polymorphism (SNP) sites (relative to the full-length hutC sequence)
456459478481482498506523546569579647
B. cepaciaCTCGTGATGGGT
B. cenocepaciaCCGGCGATCGGT
B. arborisCCCGCGTTTCGT
B. seminalisCCCACGATCTGT
B. pyrrociniaCTCACGATCGGT
B. stabilisMCCACGTTCGGT
B. metallicaCCCACGATGGGT
B. aenigmaticaCTCGTGATGGAT
B. lataCYCGTGATGGGA
B. contaminansCCCGTGTTGGGA
B. purquaeCCCGTAAGGGGT
B. paludisCTCGTGTGCGCT
B. pseudomultivoransGTCACATTCTGT
B. diffusaMACGCATTGGGT
B. territoriiCAGGTATTGGGT
B. ambifariaCACGCATTCGGT
B. anthinaGACACATTCGGT
B. latensGACACATTGGGT
B. dolosaCACGCGATCGGT
B. multivoransCMMCAGAGCCGT
B. vietnamiensisCTGGTATCCGAT
B. stagnalisCCGCAGATGAGT
B. ubonensisGCCRGGASCAGT
B. catarinensisCCCGCGAGGGGT
B. solaCTCACGACCGGT
B. semiaridaCCCACGAGCGGT
B. orbicolaCCCACGATCGGT
), ArticleFig(id=1217784601842467183, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=EN, label=Table 6, caption=

Statistics of differential base numbers among Bcc species in the characteristic barcode of hutC gene sequence

, figureFileSmall=null, figureFileBig=null, tableContent=
NumberName of Bcc species123456789101112131415161718192021222324252627

总数

Total

1B. cepacia/
2B. cenocepacia4/
3B. arboris44/
4B. seminalis544/
5B. pyrrocinia3322/
6B. stabilis53322/
7B. metalica334222/
8B. aenigmatica1565464/
9B. lata14554532/
10B. contaminans354664441/
11B. purquae3566664434/
12B. paludis46676574555/
13B. pseudomultivorans756344688887/
14B. diffusa4544544554465/
15B. territorii45677665544672/
16B. ambifaria544543566555413/
17B. anthina7665435887772353/
18B. latens67667447766832431/
19B. dolosa324323344555635245/
20B. multivorans6554555767568786784/
21B. vietnaminans66897795776476457889/
22B. stagnalis546566465669976898649/
23B. ubonensis6564555767566897665386/
24B. catarinensis33444424342584657634854/
25B. sola446313355765568556356744/
26B. semiarida5352222656456685663386322/
27B. orbicola42411115455655744524854321/
总数Total111112128113104103102133122134130150155119147118142145102146182163146111120112983 448
均值Average value4.34.34.94.34.04.03.95.14.75.25.05.86.04.65.74.55.55.63.95.67.06.35.64.34.64.33.84.91
), ArticleFig(id=1217784601938936181, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800732894572624, language=CN, label=表6, caption=

Bcc各个种之间在特征碱基条码中的差异碱基数目统计

, figureFileSmall=null, figureFileBig=null, tableContent=
NumberName of Bcc species123456789101112131415161718192021222324252627

总数

Total

1B. cepacia/
2B. cenocepacia4/
3B. arboris44/
4B. seminalis544/
5B. pyrrocinia3322/
6B. stabilis53322/
7B. metalica334222/
8B. aenigmatica1565464/
9B. lata14554532/
10B. contaminans354664441/
11B. purquae3566664434/
12B. paludis46676574555/
13B. pseudomultivorans756344688887/
14B. diffusa4544544554465/
15B. territorii45677665544672/
16B. ambifaria544543566555413/
17B. anthina7665435887772353/
18B. latens67667447766832431/
19B. dolosa324323344555635245/
20B. multivorans6554555767568786784/
21B. vietnaminans66897795776476457889/
22B. stagnalis546566465669976898649/
23B. ubonensis6564555767566897665386/
24B. catarinensis33444424342584657634854/
25B. sola446313355765568556356744/
26B. semiarida5352222656456685663386322/
27B. orbicola42411115455655744524854321/
总数Total111112128113104103102133122134130150155119147118142145102146182163146111120112983 448
均值Average value4.34.34.94.34.04.03.95.14.75.25.05.86.04.65.74.55.55.63.95.67.06.35.64.34.64.33.84.91
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利用 hutC 基因对洋葱伯克霍尔德菌群种水平的鉴定探讨
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余萌 1 , 王似锦 1 , 张迅迪 1, 2 , 马仕洪 1, *
微生物学报 | 研究报告 2025,65(12): 5339-5351
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微生物学报 | 研究报告 2025, 65(12): 5339-5351
利用 hutC 基因对洋葱伯克霍尔德菌群种水平的鉴定探讨
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余萌1, 王似锦1, 张迅迪1, 2, 马仕洪1, *
作者信息
  • 1.中国食品药品检定研究院，北京
  • 2.中国药科大学，江苏 南京
Species-level identification of Burkholderia cepacia complex through hutC sequence analysis
Meng YU1, Sijin WANG1, Xundi ZHANG1, 2, Shihong MA1, *
Affiliations
  • 1.National Institutes for Food and Drug Control, Beijing, China
  • 2.China Pharmaceutical University, Nanjing, Jiangsu, China
出版时间: 2025-12-04 doi: 10.13343/j.cnki.wsxb.20250354
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摘要
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目的 探讨利用hutC基因对洋葱伯克霍尔德菌群(Burkholderia cepacia complex, Bcc)进行种水平鉴定的可行性。 方法 对hutC基因序列及其编码蛋白的氨基酸序列进行比对和系统发育分析，从理论上评估用于Bcc种水平鉴定的可行性。针对Bcc的hutC基因设计该基因扩增引物，以标准菌株为模板扩增Bcc代表菌种的hutC基因，将测序结果与NCBI序列进行比对和系统发育分析以验证前述理论假设。此外，对hutC基因序列进行单核苷酸多态性分析，查找Bcc种水平的特征条码。 结果 hutC基因在Bcc和非Bcc菌种间相对保守。采用所设计的引物和扩增条件可扩增出12株Bcc标准菌株长度为692 bp的hutC基因序列。除1株分类学有误的菌株外，其余11株的比对结果均与保藏中心一致。对hutC基因的系统发育分析结果表明，不同Bcc菌种能够以较高的自展值聚类。由12个核苷酸组成的hutC基因特征条码可快速区分不同的Bcc菌种。 结论 hutC基因可作为Bcc鉴定的新型看家基因靶点，用于Bcc菌群和菌种水平的准确鉴定。

洋葱伯克霍尔德菌群  /  hutC基因  /  看家基因  /  微生物鉴定

Objective To explore the application of hutC sequence analysis in the identification of Burkholderia cepacia complex (Bcc) at the species level. Methods We compared the sequences of hutC and the amino acid sequences of the encoded proteins and conducted phylogenetic analysis to theoretically assess the feasibility of using hutC for species-level identification of Bcc. Primers targeting the hutC of Bcc were designed, and the amplification conditions were optimized. With those of standard strains as templates, the hutC sequences of representative Bcc species were amplified. The sequencing results were compared with NCBI sequences for phylogenetic analysis to validate the theoretical hypothesis. Additionally, single nucleotide polymorphism analysis of hutC sequences was performed to identify species-level characteristic barcodes of Bcc. Results The hutC gene was relatively conserved between Bcc and non-Bcc. With the designed primers and amplification conditions, a 692 bp fragment of hutC was successfully amplified from 12 Bcc standard strains. Except for one strain with taxonomic errors, the remaining 11 strains had the comparison results consistent with those from the culture collection centers. The phylogenetic analysis based on hutC showed that different Bcc species could be clustered with high bootstrap values. A 12 nucleotide characteristic barcode of hutC was identified, which can rapidly distinguish different species of Bcc. Conclusion The gene hutC can serve as a new housekeeping gene target for accurate identification of Bcc at the complex and species levels.

Burkholderia cepacia complex  /  hutC  /  housekeeping gene  /  microbial identification
余萌, 王似锦, 张迅迪, 马仕洪. 利用 hutC 基因对洋葱伯克霍尔德菌群种水平的鉴定探讨. 微生物学报, 2025 , 65 (12) : 5339 -5351 . DOI: 10.13343/j.cnki.wsxb.20250354
Meng YU, Sijin WANG, Xundi ZHANG, Shihong MA. Species-level identification of Burkholderia cepacia complex through hutC sequence analysis[J]. Acta Microbiologica Sinica, 2025 , 65 (12) : 5339 -5351 . DOI: 10.13343/j.cnki.wsxb.20250354
正文
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洋葱伯克霍尔德菌群(Burkholderia cepacia complex, Bcc)隶属于伯克霍尔德菌属(Burkholderia)，由若干表型近似但基因型存在差异的菌种组成，其代表菌种为洋葱伯克霍尔德菌(Burkholderia cepacia)。B.cepacia于1950年首次在腐烂的洋葱根茎中被发现，最初被命 名为洋葱假单胞菌(Pseudomonas cepacia)[1]。1992年，Yabuuchi等[2]通过多相分类学方法依据16S rRNA基因序列、DNA-DNA杂交同源性等将假单胞菌属rRNA杂交II组定义为伯克霍尔德菌属。1997年，Vandamme等对128株伯克霍尔德菌属菌株进行了系统的表型和基因型分析[3]，这些被鉴定为“B. cepacia”的若干菌株通过DNA-DNA杂交又进一步被划分为至少5个不同的基因型亚群(genotypic subgroups)。同年5月，在洋葱伯克霍尔德菌国际工作组第3次会议(third meeting of the international Burkholderia cepacia working group, May 10 to 11, 1997, Victoria, British Columbia, Canada)上提议将上述表型特征近似的基因型亚群命名为Bcc，并针对其分类学特点采用“基因型(genomovar)”对不同亚群进行命名，至此，Bcc被分为5个基因型，分别是基因型I (对应种名：B. cepacia)、基因型II (B. multivorans)、基因型III (B. cenocepacia)、基因型IV (B. stabilis)、基因型V (B. vietnamiensis)。随着分子生物学分析技术的飞速发展，基因水平的信息为Bcc的准确分类提供了新的依据，多位点序列分型、全基因组测序等技术在Bcc分类学研究中的应用使得Bcc家族不断有新的成员加入，截至2024年，已被分类命名的Bcc成员达到了27个种[4-6]。
Bcc是环境中广泛存在的人类条件致病菌，具有寡养、代谢谱广、天然耐药等特点，易污染药品生产水系统，且临床治疗难度大[7-9]。自1980年美国某吸入剂生产企业因去离子水制备系统控制失败导致产品污染B. cepacia，进而引发数名囊性纤维化(cystic fibrosis, CF)患者死亡事件以来，该菌一直受到美国食品和药品监督管理局(Food and Drug Administration, FDA)的关注[8]。近年来，非无菌水基质药品因存在Bcc污染风险而频繁被FDA召回[10-11]，FDA已明确将其列为不可接受微生物(objectionable microorganisms, OM)。美国注射剂协会(Parenteral Drug Association, PDA)第67号技术报告中指出，至少满足以下特点之一的微生物即为不可接受微生物：(1) 可在药品中增殖，改变药品理化性质，对其功能或疗效产生不利影响；(2) 在药品中的数量、致病性会导致特定给药途径下的用药患者发生感染(https://www.pda.org/bookstore/product-detail/2456-tr-67-exclusion-of-objectionable-microorganisms)。美国药典(US Pharmacopeia, USP) 42版第2增补本和《中国药典》2025年版先后收载了基于选择和分离培养的Bcc检查法[12-13]，规定了对样品是否污染Bcc进行定性判断的检查方法。
按照《中国药典》通则9204微生物鉴定指导原则的要求[14]，对于Bcc检查结果判断需达到的鉴定水平为菌群，但在实际应用中当产品发生污染或环境发生严重异常时，为确定污染源需对检出的Bcc菌株进行菌种水平的准确鉴定以便进行溯源分析。然而，由于Bcc在表型、16S rRNA基因序列等方面具有高度的相似性，使用传统的生化鉴定、基质辅助激光解析电离飞行时间质谱技术(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS)和16S rRNA基因序列分析均无法实现种水平的准确鉴定[15]，通常需要辅助看家基因(housekeeping gene)的序列分析，recA、hisA和gyrB等基因已被用于Bcc种水平的鉴定[16-18]。
组氨酸利用阻遏物C (histidine utilization repressor C, HutC)是革兰氏阴性细菌组氨酸代谢中特异性的转录调控因子，属于GntR蛋白家族[19]。hutC基因编码的蛋白质，其N端的DNA结合结构域较为保守，C端的效应物结合结构域差异较大[20]。这种特性使其在系统发育分析中具有潜在的应用价值，但目前尚无利用hutC基因对Bcc进行种水平鉴定的报道。本研究对Bcc的hutC基因及其编码蛋白的氨基酸序列进行分析，探讨其在Bcc与非Bcc间以及Bcc种水平成员间的保守性，并设计引物进行验证，探讨利用hutC基因进行Bcc种水平鉴定的可行性，以便在污染溯源调查时快速准确地确定污染源。
1 材料与方法
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1.1 菌株
本研究共使用12株Bcc标准菌株(表1)，分别购自美国典型培养物保藏中心(American Type Culture Collection, ATCC)、中国普通微生物菌种保藏管理中心(China General Microbiological Culture Collection Center, CGMCC)、中国工业微生物菌种保藏管理中心(China Center of Industrial Culture Collection, CICC)以及中国医学细菌保藏管理中心(China Center for Medical Bacterial Culture Collection, CMCC)。
1.2 主要试剂和仪器
胰酪大豆胨液体培养基(soybean-casein digest broth, TSB)，Merck KGaA公司；胰酪大豆胨琼脂培养基(soybean-casein digest agar, TSA)，BD医疗公司；蒸馏水，英潍捷基(上海)贸易有限公司；IFA接种液、GENIII反应板，Biolog公司；Ex Taq® DNA Polymerase，宝日医生物技术(北京)有限公司；prepGEM细菌基因组快速提取试剂盒，MicroGEM公司；引物由北京诺赛基因组研究中心有限公司合成。
电子天平，Sartorius公司；生物安全柜，NuAire公司；生化培养箱、高压灭菌器，Yamato Scientific公司；自动微生物鉴定系统，Biolog公司；基因扩增仪，Bio-Rad公司。
1.3 菌株培养和基因组提取
将Bcc菌株划线接种于TSA培养基，33 ℃培养18-24 h，按照prepGEM细菌基因组快速提取试剂盒的说明书进行基因组提取。
1.4 生化鉴定
按照Biolog自动微生物鉴定系统使用说明，挑取菌落至IFA接种液中，混匀制成规定浓度的菌悬液，按要求接种至GENIII反应板，置于自动微生物鉴定系统中培养并读取结果。
1.5 序列分析
16S rRNA基因扩增采用引物27f (5′-AGA GTTTGATCCTGGCTCAG-3′)和1492r (5′-TAC GGCTACCTTGTTACGACTT-3′)。PCR反应体系(50 μL)：Ex Taq® DNA Polymerase (5 U/μL) 0.25 μL，上、下游引物(10 μmol/L)各1 μL，10×PCR buffer 5 μL，dNTP mixture (各2.5 mmol/L) 4 μL，DNA模板1 μL，余下用ddH2O补齐。PCR反应条件：95 ℃预变性4 min；95 ℃变性30 s，50 ℃ 退火45 s，72 ℃延伸1 min 30 s，共35个循环；72 ℃终延伸5 min。recA扩增反应体系各组分与16S rRNA基因扩增体系相同，上、下游引物分别为BCR1 (5′-TGACCGCCGAG AAGAGCAA-3′)和BCR2 (5′-CTCTTCTTCGTC CATCGCCTC-3′)。反应条件：94 ℃预变性3 min；94 ℃变性30 s，56 ℃退火45 s，72 ℃延伸1 min，共32个循环；72 ℃终延伸10 min。测序扩增产物后，将序列上传至NCBI进行比对。
1.6 Bcc组氨酸利用阻遏物氨基酸序列系统发育分析
以ATCC 25416的HutC氨基酸序列(登录号为WP_175830369.1)在NCBI中进行BLASTp比对，获取Bcc其他成员种的组氨酸利用阻遏物氨基酸Refseq序列，同时从NCBI下载非Bcc近缘种的组氨酸利用阻遏物蛋白Refseq序列。将这些序列置于MEGA 12中，采用ClustalW算法进行氨基酸序列比对，使用bootstrap法检验(检验次数为1 000)，进化模型选用Jones-Taylor-Thornton，构建系统发育最大似然树。
1.7 Bcc菌株 hutC 基因序列测定
根据NCBI中Bcc的hutC基因738 bp全长序列设计扩增引物(表2)，扩增片段长度分别为702 bp (P1、P2)和692 bp (P3、P4)。反应体系(50 μL)：Ex Taq® DNA Polymerase (5 U/μL) 0.25 μL，上、下游引物各1 μL (10 μmol/L)，10×PCR buffer 5 μL，dNTP mixture (各2.5 mmol/L) 4 μL，DNA模板1 μL，ddH2O补齐。用ATCC 25416和ATCC BAA-245基因组进行反应程序的考察。PCR反应条件：95 ℃预变性5 min；95 ℃变性10 s，退火温度分别为48.0、48.5、49.3、50.7、52.3、53.6、54.5、55.0 ℃，退火时间30 s，72 ℃延伸1 min，30个循环；72 ℃终延伸5 min。使用1%琼脂糖凝胶电泳对产物进行大小和纯度检测。确定退火温度后，以上述Bcc不同菌株的基因组作为扩增模板扩增其hutC基因，将扩增产物进行测序后的序列上传至NCBI进行比对。
1.8 hutC 基因核苷酸序列用于Bcc种水平鉴定的验证
从NCBI下载Bcc不同种水平代表菌株的hutC基因序列，包括不同种的模式菌株，与上述标准菌株的hutC序列一起置于MEGA 12中，采用ClustalW算法进行核苷酸序列比对，使用bootstrap法检验(检验次数为1 000)，进化模型选用maximum composite likelihood，构建系统发育树。
在此基础上，进一步分析Bcc不同种的hutC基因序列，在MEGA 12中找出27个种水平成员种内的共识序列(consensus sequence)，随后将这些共识序列进行比对，获取多态性位点的信息，从而寻找可用于Bcc种水平区分的位点。
2 结果与分析
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2.1 标准菌株的鉴定结果
采用常规细菌鉴定方法对Bcc标准菌株进行鉴定(表3)。recA序列分析基本能将Bcc标准菌株准确鉴定至菌种水平，CMCC(B) 23010的序列比对结果为B. lata (98.40%)。CGMCC 1.3816的比对结果为B. ambifaria，与保藏中心给出的B. pyrrocinia不符，推测原因可能是保藏中心未能依据分类学变化及时更新菌种名称。Biolog生化鉴定将CGMCC 1.10511鉴定为假单胞菌属，其他标准菌株仅能鉴定至Bcc菌群水平，其中ATCC BAA-245、ATCC BAA-246、ATCC BAA-247、CICC 23882、CGMCC 1.2982可鉴定至种水平。16S rRNA基因序列分析均能将标准菌株鉴定至Bcc菌群水平，但由于Bcc菌种间的16S rRNA基因序列相似性较高，无法准确鉴定至种水平。
2.2 对Bcc组氨酸利用阻遏物氨基酸序列的系统发育分析
对Bcc种成员及其近缘菌种(Burkholderia中的非Bcc种以及Paraburkholderia种成员代表)的HutC蛋白氨基酸序列进行系统发育分析，构建最大似然树(图1)并计算遗传距离。Bcc种成员间HutC蛋白氨基酸序列的一致性为85.72%-99.54%，而Bcc与非Bcc HutC蛋白的氨基酸序列一致性远低于85.00%。系统发育分析结果表明，Bcc种水平成员聚类至同一进化分支的bootstrap值较高，提示HutC蛋白在Bcc种成员间相对保守，其编码基因可作为区分Bcc与非Bcc的靶标。
2.3 对 hutC 基因扩增引物和扩增条件的考察
使用表2中的引物对P1、P2对ATCC 25416的hutC基因进行PCR扩增，电泳检测时均可在相应位置获得特异性扩增条带，确定退火温度为54.0 ℃。在该退火温度下对表1中的试验菌株进行hutC基因PCR扩增，ATCC BAA-245和CMCC(B) 23006无法获得稳定的特异性扩增(数据未展示)。使用引物对P3、P4对ATCC BAA-245的hutC基因进行PCR扩增，由电泳检测结果可知(图2)，引物对P3在退火温度为48.0-55.0 ℃时，随着温度升高，除在目标片段大小处有扩增外，在大于2 000 bp处存在一条非特异性扩增条带；引物对P4在退火温度为50.7、49.3、48.5、48.0 ℃时，除在目标片段大小处有扩增外，在2 000 bp和500 bp左右存在非特异性扩增，当退火温度继续升高，非特异性扩增逐渐减弱，直至无明显扩增条带。因此，在后续试验中选择55.0 ℃作为hutC基因的PCR退火温度。使用引物对P4，以表1中的Bcc菌株作为模板进行hutC基因扩增，扩增产物电泳结果如图3所示，各试验菌株在700 bp大小附近均呈现特异性扩增。
2.4 对 hutC 基因应用于Bcc种水平鉴定的验证
将扩增后的hutC基因序列在NCBI进行比对(表4)，12株标准菌株中有11株与保藏中心提供的种水平名称一致，且比对相似度均大于99.00%，说明hutC序列对Bcc种水平的鉴定结果准确；CGMCC 1.3816的比对结果为B. ambifaria，与recA鉴定结果一致。
将测序所得试验菌株的hutC基因序列与NCBI中Bcc 27个菌种共80株菌株的hutC序列共同构建系统发育邻接树，每个菌种均选取其模式菌株(type strain)，另根据数据库实际情况选择同种水平的其他非模式菌株(图4)。通过遗传距离计算，不同Bcc种间相似性最大为97%，种内相似性为98%-100%。系统发育树表明，Bcc同种水平的不同菌株均以较高的自展值(bootstrap value)聚类至同一分支，不同种水平间区分良好。自测获得的12株Bcc试验标准菌株的hutC序列也均可聚类至相应的种水平分支中。上述结果表明，hutC基因序列在Bcc种内保守性较好，可应用于Bcc的种水平鉴定。当进行溯源分析时，在完成种水平的鉴定后通过进一步对比污染菌的hutC基因序列，如和目标菌的序列不同则可以排除其为污染源；如和目标菌的序列相同，虽不能肯定其为污染源，但为进一步的溯源分析明确了方向。
2.5 hutC 基因序列中用于Bcc种水平鉴定的特征碱基条码
鉴于hutC基因可作为Bcc种水平鉴定的一个有效看家基因靶点，通过对Bcc种水平菌株hutC基因的比对可寻找出特征性位点，实现在不构建系统发育树的情况下识别Bcc的不同种。
按照1.8节方法检测出hutC基因的211个多态性位点，对其中具备种水平区分能力的位点进行分析，最终筛选出12个位点，它们包含在hutC序列192 bp区域内，分别位于位点456、459、478、481、482、498、506、523、546、569、579、647 (表5)。这些位点上的核苷酸组合成一个12位的碱基代码，从而能够区分27种Bcc。表6对Bcc不同种在这12个碱基组成的特征条码中的碱基差异进行了统计，结果表明27个种种间的碱基总差异数目为3 448个，每个物种之间的平均错配数为4.91 (范围在3.80-7.00)，这表明即便未来会有新的Bcc种成员被发现和命名，这12个核苷酸组成的特征条码仍然在较大概率上对Bcc有较好的种水平区分效果。
3 讨论与结论
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目前，《中国药典》和USP对Bcc的检查结果判断时的鉴定要求均为菌群水平，商品化的鉴定方法如生化法和MALDI-TOF MS对于Bcc菌种水平的鉴定效果不佳[21-23]。虽然通常16S rRNA基因序列分析被认为是细菌鉴定的“金标准”，但由于Bcc种间16S rRNA基因序列的相似性在98.3%-99.3%之间[23]，16S rRNA基因序列分析也无法对Bcc进行准确的种间区分。目前使用看家基因的序列分析和限制性片段长度多态性分析通常可得到较准确的Bcc种水平鉴定结果[17]。例如，应用最为广泛的recA基因，有文献报道其在Bcc成员间的相似值为94.2%-95.6%，而种内的相似值为98.0%-99.0%[24]。然而随着Bcc分类的发展，研究者发现部分种的recA序列相似性较高，仅依靠recA序列分析获得准确的种水平鉴定存在一定难度[25]。
hutC基因于20世纪70年代首次在肠道细菌中被发现，第一个hutC基因的完整核苷酸序列是由Allison等于1990年从恶臭假单胞菌(Pseudomonas putida)中获得[26]。目前，对细菌中hutC基因的研究主要集中在其功能性方面。hutC基因除了调节组氨酸的代谢外，还与细菌毒力、生物膜形成、氮源代谢以及运动性等功能有关[20,27]。对伯克霍尔德菌属的分子信标和系统发育研究发现，Bcc的hutC基因相对非Bcc有2个碱基的插入[28]，尚未见有利用hutC基因进行Bcc种水平鉴定的研究。本研究首先对hutC基因的编码蛋白进行了系统发育分析，证明其在Bcc菌群与非Bcc间相对保守；在此基础上设计了hutC基因的扩增引物，考察并优化了PCR反应条件，以标准菌株为模板，对12株Bcc菌株的hutC基因进行了扩增、测序；并将测序结果与从NCBI下载的27个种的Bcc代表菌株的hutC基因序列进行系统发育分析，证明hutC基因对Bcc种成员间的区分效果良好，利用hutC基因可实现对Bcc种水平的准确鉴定。在上述工作的基础上，通过对Bcc hutC基因单核苷酸多态性位点的分析最终选择序列中部靠近3′端的12个核苷酸位点组成了Bcc种水平特征条码，可用于Bcc的快速种水平确定。
目前，USP和《中国药典》收载的Bcc检查法在结果判断环节均要求进行菌群水平的确认。通过常规鉴定方法，如MALDI-TOF MS、16S rRNA基因序列分析等可以实现此目标。当对生产过程和终产品中污染的Bcc进行溯源调查时则需要对污染微生物进行菌种乃至菌株水平的鉴定。应用单一看家基因的序列分析或者联合多基因位点序列分析能够满足相应要求。本研究证明，hutC基因可作为Bcc种水平鉴定的新的看家基因靶点，可应用于药品终产品和生产过程中Bcc污染溯源时种水平的准确鉴定。
作者贡献声明
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余萌：数据收集和分析，实验验证，论文撰写；王似锦：论文修改，分析指导；张迅迪：部分实验验证，论文修改；马仕洪：分析指导，论文审阅，项目管理。
作者利益冲突公开声明
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作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。
基金
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  • 中国食品药品检定研究院化学药品检定所重点实验室项目(2025HYZX04)
文献
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2025年第65卷第12期
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doi: 10.13343/j.cnki.wsxb.20250354
  • 接收时间：2025-04-29
  • 首发时间：2025-12-08
  • 出版时间：2025-12-04
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  • 收稿日期：2025-04-29
  • 录用日期：2025-06-12
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NIFDC Key Laboratory of Chemical Drug Analysis Project(2025HYZX04)
中国食品药品检定研究院化学药品检定所重点实验室项目(2025HYZX04)
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    1.中国食品药品检定研究院，北京
    2.中国药科大学，江苏 南京

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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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