WO2012068956A1 - Kit de détection pour pathogènes bactériens dans des échantillons d'urine et méthode de détection de ces derniers - Google Patents

Kit de détection pour pathogènes bactériens dans des échantillons d'urine et méthode de détection de ces derniers Download PDF

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WO2012068956A1
WO2012068956A1 PCT/CN2011/081979 CN2011081979W WO2012068956A1 WO 2012068956 A1 WO2012068956 A1 WO 2012068956A1 CN 2011081979 W CN2011081979 W CN 2011081979W WO 2012068956 A1 WO2012068956 A1 WO 2012068956A1
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seq
buffer
detecting
kit
bacterial
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PCT/CN2011/081979
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English (en)
Chinese (zh)
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任绪义
吕江峰
严一红
虞闰六
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杭州迪安医学检验中心有限公司
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Publication of WO2012068956A1 publication Critical patent/WO2012068956A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the invention belongs to the technical field of clinical microbial identification, and particularly relates to a kit for detecting bacterial pathogens in urine samples and a detection method thereof. Background technique
  • Urinary tract infections worldwide are among the most common bacterial infections, often leading to severe urinary sepsis and even death, especially in immunocompromised patients. Studies have shown that about 50% of women have had at least one urinary tract infection in their lifetime. At the same time, approximately 10% of children develop urinary tract infections each year, causing severe febrile illness with symptoms such as dysuria and abdominal pain.
  • Urine microscopy is a simple and rapid method for detecting urinary pathogens, but its drawback is that it can only detect the presence or absence of pathogens in the urine and cannot identify the specific pathogen species, and its low sensitivity is also limited to a certain extent. Widely used in clinical practice.
  • the urea test is mainly used to detect the most common Enterobacteriaceae bacteria in urine, and it is impossible to detect bacteria that do not produce urea, such as the genus Monocytogenes and Enterococcus.
  • PCR-binding DNA sequencing technology has proven to be an effective means of detecting bacteria and other microorganisms.
  • 16S rRNA exists in multiple copies in all bacteria, and its structure consists of 8 conserved regions and 9 variable regions. Therefore, primers designed in the 16S rRNA conserved region can theoretically amplify most bacterial DNA fragments, and then distinguish different bacterial species by measuring the variable regions in the amplified products.
  • Pyrosequencing technology is a next-generation DNA sequence analysis technology that eliminates the need for electrophoresis and eliminates the need for fluorescent labeling of DNA fragments for easy operation.
  • Pyrosequencing technology is an enzyme cascade chemiluminescence reaction in the same reaction system catalyzed by four enzymes. In each round of sequencing reaction, only one dNTP is added. If the dNTP is paired with a template, the polymerase can incorporate it. Go to the primer strand and release an equimolar amount of pyrophosphate groups (PPi). PPi can eventually be converted to a visible light signal and converted to a peak by PyrogramTM. Each peak The height of the book value is proportional to the number of nucleotides incorporated in the reaction.
  • Monstein et al. used pyrosequencing to detect the VI and V3 sequences of 16S rDNA gene of Helicobacter pylori 16S rDNA gene.
  • the 23 Helicobacter pylori specimens obtained from the gastroscopic biopsy specimens were identified based on the nucleotide sequence differences of the VI region. Different allele types, except for 11 samples that are consistent with the sequence of Helicobacter pylori 26 695 strains, one sample is consistent with the sequence of 199 strains, and there are 1 or 2 nucleotides according to the change of the VI region. Mutations or single nucleotide insertions are divided into 4 allelic types.
  • Martin et al used this technique to identify the ptxS1 gene related to the virulence of Bordetella pertussis, and compared it with quantitative PCR. The results showed that the two methods have good consistency.
  • the pyrosequencing technology is suitable for B. pertussis. Large-scale screening.
  • Alistair et al. used pyrosequencing to detect the anti-linezolid site on the 23s rRNA of Enterococcus, and compared with the PCR-RFLP method, the results showed 100% agreement. Marjo et al.
  • the kit uses the design software to sequence and combine the 25 bp sequence of common clinical bacterial sequencing primers to generate a pyrosequencing virtual pattern database for multiple bacterial infections.
  • the urine samples are amplified by DNA, amplified by universal primers, and then used. Universal primers were used as sequencing primers, and the sequencing results were compared with the pyrosequencing virtual pattern database.
  • the method can be used to distinguish common infections of common bacteria and different bacteria at one time, and can be used for clinical diagnosis and drug resistance monitoring of bacterial infections.
  • the blank control used can effectively deduct the possible pollution in the type judgment.
  • the specification solves the high sensitivity that inevitably leads to difficult pollution problems.
  • the technical problem to be solved by the present invention is to provide a kit for detecting a bacterial pathogen of a urine sample.
  • the technical problem to be solved by the present invention is to provide a detection method of the above kit.
  • a kit for detecting bacterial pathogens in urine samples which comprises:
  • a universal primer for amplifying the VI variable region of the 16S rR A of the bacterium a nucleotide sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 2;
  • Sequencing primer the nucleotide sequence thereof is shown as SEQ ID NO: 2;
  • SEQ ID NO: 1 marks biotin at its 5' end
  • the kit for rapid identification of the above bacteria specifically includes the following reagents:
  • DNA extraction reagent 50 mg/mL Chelex 100 buffer, 20 mg/mL proteinase K;
  • Reaction solution PCR Buffer, universal primer SEQ ID NO: 1-2, 2 mM MgCl 2 , 0.2 mM dNTPs, 2 ⁇ ]/ ⁇ Taq DNA polymerase;
  • the PCR Buffer is specifically: 0.1% (v / v) NP-40, 0.02% (v / v) gelatin, 0.6mg / mL BSA, 0.1% (v / v) Tween-20, 0.06M pH8.9 Tricine .
  • the binding buffer is specifically: 10 mM Tris-HCl, 2 ⁇ NaCl, 1 mM EDTA, 0.1% (v/v) Tween-20; the annealing buffer is specifically: 20 mM pH 7.6 Tris-Acetate, 2 mM magnesium acetate.
  • Sequencing reagents DNA polymerase, ATP sulfated enzyme, luciferase, adenosine triphosphatase, substrate APS, fluorescein and dNTP (dNTP ⁇ dATP S, dTTP, dCTP, dGTP).
  • kits for rapid identification of bacteria in a urine sample which comprises the base complementary sequence of all nucleotide sequences described above
  • the description of the book which includes:
  • a universal primer for amplifying a VI variable region of the 16SrR A a nucleotide sequence as shown in SEQ ID NO: 3 and SEQ ID NO: 4;
  • Sequencing primer the nucleotide sequence thereof is shown as SEQ ID NO: 4.
  • SEQ ID NO: 3 marks biotin at its 5' end
  • the kit for rapid identification of the above bacteria specifically includes the following reagents:
  • DNA extraction reagent 50 mg/mL Chelex 100 buffer, 20 mg/mL proteinase K;
  • Reaction solution PCR Buffer, universal primer SEQ ID NO: 3-4, 2 mM MgCl 2 , 0.2 mM dNTPs, 2 ⁇ ]/ ⁇ Taq DNA polymerase;
  • PCR Buffer is specifically: 0.1% (v / v) NP-40, 0.02% (v / v) gelatin, 0.6 mg / mL BSA, 0.1% (v / v) Tween-20, 0.06M pH8.9 Tricine .
  • the binding buffer is specifically: 10 mM Tris-HCl, 2 ⁇ NaCl, 1 mM EDTA, 0.1% (v/v) Tween-20; the annealing buffer is specifically: 20 mM pH 7.6 Tris-Acetate, 2 mM magnesium acetate.
  • Sequencing reagents DNA polymerase, ATP sulfated enzyme, luciferase, adenosine triphosphatase, substrate APS, fluorescein and dNTP (dNTP ⁇ dATPS, dTTP, dCTP, dGTP).
  • the bacteria according to the present invention are narrowly defined bacteria, including: Pseudomonas, Staphylococcus, Enterococcus, Streptococcus, Klebsiella, Citrobacter, Proteus, Acinetobacter Genus.
  • the above method for detecting a rapid test kit for urine samples includes the following steps:
  • the PCR amplification system is: lOxPCR buffer 5 L, SEQ ID NO: 10.3 L, Instruction manual
  • SEQ ID NO: 2 0.3 ⁇ , Template DNA 3 ⁇ , 0.2 mM dNTPs, 2 mM MgCl 2 , Taq DNA polymerase 2 U, add sterile water to 50 ⁇ , PCR amplification conditions are: 94 °C 3 min; 28 cycles ( 94 °C 25s, 42 °C 30s, 72 °C 20s); 72 °C 3min.
  • SEQ ID NO: 3 is substituted for SEQ ID NO: 1
  • SEQ ID NO: 4 is substituted for SEQ ID NO: 2 above, and other PCR amplification systems and conditions are the same.
  • the kit of the present invention utilizes the sequence and database alignment of the positive segment of the VI determined by pyrosequencing to determine the bacterial species.
  • This kit can be used to identify bacterial species in urine samples at one time. It can be used for the clinical diagnosis of bacterial pathogens in urine samples. It not only wins valuable rescue time for clinical diagnosis and treatment, but also has the advantages of low cost, convenient operation and strong specificity.
  • the method of the invention can greatly shorten the identification time of the bacterial species of the urine sample, and shortens from the conventionally required 24-48 hours to 5 hours, and the cost is one-third of the traditional identification method.
  • the identification of the bacteria by the sequence alignment is The ultimate method for the identification of strains, especially for some difficult to identify bacteria, the sequence alignment identification is more advantageous.
  • Figure 1 shows the results of detection of pathogenic bacteria in urine samples of a patient.
  • the sequencing result is GTAACAGGAA GAAGCTTGCT TC, which is compared with the database to determine Escherichia coli.
  • Figure 2 is a graph showing the results of detection of pathogenic bacteria in urine samples of a patient.
  • the sequencing result is GAATCCAGGA GCAAGCTCCC TTCATCCG, which is determined to be Pseudomonas aeruginosa compared with the database.
  • Figure 3 is a graph showing the results of detection of pathogenic bacteria in urine samples of a patient.
  • the sequencing result is AACGTCAGAG GAGCAAGCTC CTCG, which is compared with the database and identified as Staphylococcus epidermidis.
  • Figure 4 is a graph showing the results of detection of pathogenic bacteria in urine samples of a patient.
  • the sequencing result is CGTCACCCGA GAGCAAGCTC TC, which is compared with the database and determined to be Klebsiella pneumoniae.
  • Figure 5 is a graph showing the results of detection of pathogenic bacteria in urine samples of a patient.
  • the sequencing result is CGTCAGCAAG AAAGCAAGCT TTC, which is compared with the database and determined to be Proteus mirabilis.
  • Figure 6 is a graph showing the results of detection of pathogenic bacteria in urine samples of a patient.
  • the sequencing results were determined to be a mixed infection of Enterococcus faecium and Stenotrophomonas maltophilia.
  • Figure 7 is a graph showing the results of detection of pathogenic bacteria in urine samples of a patient.
  • the sequencing result is CCTCTTTTCC GGTGGAGCAA GCTCCGGTGG, which is determined to be Enterococcus faecium compared with the database.
  • Figure 8 is a graph showing the results of detection of pathogenic bacteria in urine samples of a patient.
  • the sequencing result is CCTCTTTCCA.
  • Example 1 Method for preparing a kit.
  • Chelex 100 Buffer Contains 0.3 mg/mL SDS (purchased from Hangzhou Fluorescent Biotech Co., Ltd.), l% (v/v) Tween-20 (purchased from Sigma, USA), 1% (v/v) NP-40 (purchased from Sigma, USA)
  • PCR Buffer 0.1% (v/v) NP-40 (purchased from Sigma, USA), 0.02% (v/v) gelatin (purchased from Sigma, USA), 0.06% (w/v) g/mL BSA (purchased In Sigma, USA, 0.1% (v/v) Tween-20 (purchased from Sigma, USA), 0.06M pH8.9 Tricine (purchased from Merck, Germany),
  • MgCl 2 purchased from Sigma, USA
  • 0.2 mM dNTPs purchased from Shanghai Dingguo Biotechnology Co., Ltd.
  • Tris-base was purchased from Sigma, USA, and anhydrous acetic acid was purchased from Hangzhou Chemical Reagent Co., Ltd.
  • Binding buffer 10mM Tris-HCl (Tris-base purchased from Sigma, USA, hydrochloric acid purchased from Hangzhou Chemical Reagent Co., Ltd.), 2M NaCl (purchased from Shanghai Test Sihewei Chemical Co., Ltd.), ImM EDTA (purchased in Hangzhou) Chemical Reagent Co., Ltd., 0.1% (v/v) Tween 20 (purchased from Sigma, USA),
  • Annealing buffer purchased from 20 mM Tris-Acetate (pH 7.6) (Tris-base, Sigma, USA, no Description: Water acetic acid purchased from Hangzhou Chemical Reagent Co., Ltd.), 2mM magnesium acetate (purchased in Shanghai Test Sihewei Chemical Co., Ltd.).
  • DNA polymerase ATP sulfated enzyme, luciferase, and adenosine triphosphatase: purchased from QIAGEN,
  • Substrate APS and Fluorescein purchased from QIAGEN,
  • dNTPs Four types of dNTPs (dATP S, dTTP, dCTP, dGTP): purchased from QIAGEN.
  • dATP S dATP S, dTTP, dCTP, dGTP
  • dGTP dGTP
  • Extracting bacterial DNA specifically including the following steps:
  • the PCR amplification system is: lOxPCR buffer 5 L, SEQ ID NO: 1 0.3 L, SEQ ID NO: 2 0.3 ⁇ £, Template DNA 3 ⁇ , 0.2 mM dNTPs, 2 mM MgCl 2 , Taq DNA polymerase 2U, add sterile water to 50 ⁇ ; PCR amplification conditions are: 94 °C 3min ; 28 cycles (94 °C 25s, 42 °C 30s, 72 °C 20s); 72 °C 3min; or SEQ ID NO : 3 Replace the above SEQ ID NO: 1, and replace SEQ ID NO: 4 with the above SEQ ID NO: 2, and the other PCR amplification systems and conditions are the same.
  • step (3) Combining the PCR amplification product obtained in step (2) with avidin-labeled magnetic beads for single-strand purification: • Ensure that all solutions reach room temperature before use;
  • the method of the present invention was applied to the identification of 8 clinical urine specimens, and the sequencing diagram is shown in Fig. 1-8.
  • the method of the present invention is compared with the microbial culture and the pathogenic bacteria are purified, and the results are consistent with the microbial identification system of the French bioMérieux.

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Abstract

L'invention concerne un kit d'identification rapide de bactéries dans des échantillons d'urine et une méthode prévue à cet effet. Le kit comprend deux amorces universelles utilisées pour amplifier la région variable VI de l'ARNr 16S bactérien, une amorce de séquençage utilisée pour détecter la séquence du segment positif VI, et des billes magnétiques marquées par l'avidine.
PCT/CN2011/081979 2010-11-25 2011-11-09 Kit de détection pour pathogènes bactériens dans des échantillons d'urine et méthode de détection de ces derniers WO2012068956A1 (fr)

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CN201010558217.6 2010-11-25
CN2010105582176A CN101974646A (zh) 2010-11-25 2010-11-25 尿液样本细菌性病原体检测试剂盒及其检测方法

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CN101974646A (zh) * 2010-11-25 2011-02-16 杭州迪安医学检验中心有限公司 尿液样本细菌性病原体检测试剂盒及其检测方法
CN106893760B (zh) * 2017-03-15 2021-08-06 杭州迪安生物技术有限公司 检测尿液病原菌的试剂盒及应用
CN108949920A (zh) * 2018-08-01 2018-12-07 深圳市领治医学科技有限公司 一种疾病相关的菌群定量扩增方法
CN109504790A (zh) * 2019-01-15 2019-03-22 上海交通大学医学院附属上海儿童医学中心 评价母乳性黄疸的生物标志物、试剂盒以及方法
CN110295241A (zh) * 2019-07-11 2019-10-01 深圳易致生物科技有限公司 用于检测尿路感染的引物组和包含该引物组的试剂盒

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CN101864490A (zh) * 2010-07-07 2010-10-20 杭州迪安医学检验中心有限公司 菌血症检测试剂盒及其检测方法
CN101921826A (zh) * 2010-01-27 2010-12-22 南京迪安医学检测中心有限公司 细菌快速鉴定的试剂盒及其检测方法
CN101974646A (zh) * 2010-11-25 2011-02-16 杭州迪安医学检验中心有限公司 尿液样本细菌性病原体检测试剂盒及其检测方法

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US20040023209A1 (en) * 2001-11-28 2004-02-05 Jon Jonasson Method for identifying microorganisms based on sequencing gene fragments
GB0202891D0 (en) * 2002-02-07 2002-03-27 Pyrosequencing Ab Microorganism identification
CN1296490C (zh) * 2004-05-19 2007-01-24 厦门大学 细菌微量快速同步检测方法

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Publication number Priority date Publication date Assignee Title
CN101921826A (zh) * 2010-01-27 2010-12-22 南京迪安医学检测中心有限公司 细菌快速鉴定的试剂盒及其检测方法
CN101864490A (zh) * 2010-07-07 2010-10-20 杭州迪安医学检验中心有限公司 菌血症检测试剂盒及其检测方法
CN101974646A (zh) * 2010-11-25 2011-02-16 杭州迪安医学检验中心有限公司 尿液样本细菌性病原体检测试剂盒及其检测方法

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