CN112063702A - Method for analyzing and identifying clinical problematic strain by 16S rRNA gene sequence - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for analyzing and identifying clinical problematic strains by 16S rRNA gene sequences. The method for identifying clinical problematic strains adopts a 16S rRNA gene sequence analysis method, and comprises the following steps: s1: extracting bacterial DNA of a strain to be identified, selecting a bacterial universal primer, and establishing a PCR method for amplifying bacterial 16S rRNA; s2: optimizing a PCR reaction system and reaction conditions to obtain an amplification product; s3: and comparing the amplified product with a gene database to obtain the strain of the bacteria to be identified. The invention establishes a new method for effectively identifying the difficult and complicated strains by using a 16S rRNA gene sequence analysis technology, expands the identification spectrum of clinical strains and meets the clinical identification requirement on the difficult and complicated strains.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for analyzing and identifying clinical problematic strains by 16S rRNA gene sequences.
Background
16S rRNA gene sequence analysis refers to the analysis of gene sequences by cloning, sequencing or enzyme digestion and probe hybridization16S rRNA sequence information of microorganisms in the sample is obtained through hybridization and is compared with a 16S rRNA database, so that species identification and evolutionary tree analysis are carried out on the microorganisms in the sample, and the method is a quick and effective method. 16S rRNA is present in all organisms, has evolved with good clock properties, is highly conserved in structure and function, and is called "bacteriolysis", so 16S rRNA is the most common molecular clock in bacterial species identification and phylogenetic studies[1-2]。
In biological cells, ribosomal rna (rrna) binds to several tens of proteins to form ribosomes, which then move along an mRNA template to perform a function of protein synthesis, which plays an essential role in phylogeny. In the long evolution process, rRNA molecular functions are almost kept constant, and the arrangement sequence of molecules at certain positions changes very slowly to form 'bacteriolysis'[7-9]. Through the determination of the constant sequence, the genetic relationship of species can be revealed, and clues are provided for phylogeny; rRNA has high denaturation, and species identification can be performed by determining a nucleic acid sequence characteristic to a biological species. Bacterial ribosomal RNA (rRNA) was classified according to the sedimentation coefficient, and there were three types of 5S, 16S and 23S rRNA. 16S rRNA is shared by cells, has large proportion in the content of bacterial RNA, has moderate molecular weight of about 1.5KB, can reflect the difference between different genera, and can obtain sequences easily through a sequencing technology, so that the 16S rRNA gene sequence is most suitable as the development and identification index of a biological system[10-11]. For bacteria that cannot be identified clinically by conventional methods, 16S rRNA gene sequence analysis techniques are adopted by numerous scholars such as Drancouurt and the like, which solves the puzzle[1-2,7,10-12]. The 16S rRNA gene sequence analysis technology has important clinical application value in the identification of problematic strains[13-15]。
At present, the global microbial infection shows a rapid rising trend, the drug resistance rate of bacteria is continuously increased, and newly infected pathogenic microorganisms emerge endlessly, so that higher requirements are provided for clinical microbial examination. The method for identifying various bacteria needs to be established according to self conditions in clinical microbiological laboratories, and the method needs to be fast, high in sensitivity and specificity and easy to implementAnd (5) operating. Traditional bacterial identification is mainly based on morphology and physiological properties, and requires culture and biochemical tests or immunological detection. With the development of microbiology technology, the current full-automatic bacteria identification instrument basically replaces a manual method, is convenient to operate, identifies more than 98 percent of common clinical strains, and can basically meet the initial requirements of clinical laboratories on bacteria identification. However, the following problems may also exist: the phenotype on which it depends is not stable; the sensitivity is not high, and some methods are only suitable for single strains; the utility of certain methods is also affected by serovar[3-6]. Therefore, for some rare strains, the full-automatic bacteria identifier cannot give an ideal identification result. Therefore, it is necessary to establish a new method for effectively identifying the problematic bacteria species, and to evaluate the problematic bacteria species for clinical application, so as to meet the clinical requirement for identifying the problematic bacteria species.
Disclosure of Invention
The invention aims to establish a PCR method for effectively identifying difficult and complicated strains by using a 16S rRNA gene sequence analysis technology, optimize a reaction system and conditions thereof and apply the PCR method to the identification of clinical difficult and complicated strains.
Specifically, the technical scheme of the invention is as follows:
the invention discloses a method for identifying clinical problematic strain in the first aspect, specifically, a 16S rRNA gene sequence analysis method is adopted, which comprises the following steps:
s1: extracting bacterial DNA of a strain to be identified, selecting a bacterial universal primer, and establishing a PCR method for amplifying bacterial 16S rRNA;
s2: optimizing a PCR reaction system and reaction conditions to obtain an amplification product;
s3: and comparing the amplified product with a gene database to obtain the strain of the bacteria to be identified.
It should be understood that the method of the present invention is not limited to the above steps, and those skilled in the art may add other additional steps before S1, between S1 and S2, between S2 and S3, and after S3, as needed, and all of which are within the protection scope of the present invention.
Preferably, the bacterial universal primer is a 27F and 1492R universal primer. In some embodiments of the invention, the nucleotide sequence of primer 27F is: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1); the nucleotide sequence of primer 1492R is: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2).
Preferably, the PCR reaction system comprises a primer, Pfu Buffer, dNTP, Pfu DNA polymerase, DNA template and ddH2O。
Preferably, the concentration of the primer is 0.1-0.75 mu M/L; in some embodiments of the invention, the primer concentration is 0.5. mu.M/L.
Preferably, the dosage of the Pfu DNA polymerase is 2.5-7.0U/system; in some embodiments of the invention, the Pfu DNA polymerase is used in an amount of 5.0U/system.
Preferably, the PCR reaction conditions are: pre-denaturation at 95 ℃ for 5min, followed by 35 cycles of amplification (denaturation at 95 ℃ for 30s, annealing at 52-55 ℃ for 10-40s, extension at 72 ℃ for 90s), followed by extension at 72 ℃ for 7 min.
It should be understood that the PCR reaction conditions are not limited to the above conditions, and those skilled in the art can select other suitable PCR reaction conditions according to the needs and are within the scope of the present invention.
In some preferred embodiments of the invention, the PCR annealing temperature is 52 ℃.
In some preferred embodiments of the invention, the annealing time is 30 s.
Preferably, in S3, a BLAST sequence alignment is performed using the Genebank database to obtain the species of the bacteria to be identified.
Preferably, the bacteria include baumannii holtzeri, brunella maltas and campylobacter jejuni.
In a second aspect, the invention discloses the use of a method as described above for identifying a microbial species in a sample.
On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.
Compared with the prior art, the invention has the following remarkable advantages and effects:
the invention establishes a new method for effectively identifying the difficult and complicated strains by using a 16S rRNA gene sequence analysis technology, expands the identification spectrum of clinical strains and meets the clinical identification requirement on the difficult and complicated strains.
Drawings
FIG. 1 is an agarose gel electrophoresis image of PCR products under different experimental conditions by using a 16S rRNA gene analysis method in the example of the present invention.
FIG. 2 is a diagram of bidirectional sequencing of PCR products (BioEdit Sequence Alignment Editor software analysis of fragmented sections) in an embodiment of the present invention.
FIG. 3 is a BLAST alignment of product sequences with the Genebank database in accordance with the present invention.
FIG. 4 is a BLAST alignment of product sequences with the Genebank database in accordance with the present invention.
FIG. 5 is a one-way sequencing diagram of PCR products (BioEdit Sequence Alignment Editor software analysis of fragmented sections) in an embodiment of the present invention.
FIG. 6 is a BLAST alignment of product sequences with the Genebank database in accordance with the present invention.
FIG. 7 is a diagram of bidirectional sequencing of PCR products (BioEdit Sequence Alignment Editor software analysis of fragmented sections) in an embodiment of the present invention.
FIG. 8 is a BLAST alignment of product sequences against the Genebank database in accordance with the examples of the present invention.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.
The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.
Example 1
The embodiment discloses a method for identifying clinical problematic strains, and specifically adopts a 16S rRNA gene sequence analysis method, which comprises the following steps:
s1: extracting bacterial DNA of a strain to be identified, selecting a bacterial universal primer, and establishing a PCR method for amplifying bacterial 16S rRNA;
s2: optimizing a PCR reaction system and reaction conditions to obtain an amplification product;
s3: and comparing the amplified product with a gene database to obtain the strain of the bacteria to be identified.
Specifically, the method comprises the following steps:
first, genome DNA extraction
Specimens were from bacteria that could not be identified during the years 2015 to 2019 of the university of southeast university Hospital. Activated clinical isolate 4 area was streaked onto blood plates and supplemented with 5% CO at 35 deg.C2The culture was carried out in the air for 24 to 48 hours, and then colonies were collected, and genomic DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit of Omega Bio-Tek.
Sequence analysis of di, 16S rRNA genes
The primers were diluted to 10. mu.M and PCR amplified. And (3) PCR reaction system: mu.l primer 27F, 1. mu.l primer 1492R, 5. mu.l 10 XPfu Buffer, 1. mu.l 10mmol/L dNTP, 1. mu.l 5U/. mu.l Pfu DNA polymerase, 3. mu.l DNA template, ddH2O, metering the volume to 50 mu l; and (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min, 35 cycles of amplification (denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 90s), extension at 72 ℃ for 7min, cooling the final product to 25 ℃, taking out the product for agarose gel electrophoresis analysis, recovering the target band, and sending the target band to Invitrogen for sequencing. And comparing the sequencing result with the sequence in the 16S rRNA database, and determining the position of the sequencing result in the evolutionary tree, thereby identifying the possible microorganism species in the sample.
Third, optimization of PCR system and conditions
3.1 primer concentration
In the PCR reaction system, the primer is the key of the PCR specific reaction. The yield of PCR amplification is reduced due to the fact that the concentration of the primer is too low; whereas, when the concentration of the primer is too high, a mismatch is induced, and thus non-specific amplification occurs. Therefore, 4 concentrations of 0.1, 0.2, 0.5 and 0.75. mu.M/L were set for optimization, and the concentrations of the upstream and downstream primers were identical.
3.2 amount of Pfu DNA polymerase
The PCR amplification efficiency can be improved by appropriately increasing the amount of Pfu DNA polymerase, but the occurrence of non-specific bands may be caused by excessively high amount. The amplification efficiencies were compared among the systems of 2.5, 5.0 and 7.0U/system, and the optimum concentration was selected.
3.3 PCR annealing temperature
And after denaturation, rapidly cooling to 40-60 ℃ to enable the primer and the template to be combined. The annealing temperature generally depends on the length of the primer, the base composition and its concentration, and the length of the target base sequence. Annealing temperature is a major factor affecting the specificity of PCR. Therefore, the annealing temperature was selected in the vicinity of the Tm value, and the experiment was carried out while setting 4 temperatures of 55, 54, 53 and 52 ℃.
3.4 PCR annealing time
The annealing time has a great influence on the stability of the PCR product, so that 5 times of 10, 15, 20, 30 and 40s are selected for comparison to determine the optimal annealing time.
Fourth, experimental results
When the concentration of the primer is 0.1. mu.M/L, the amplification efficiency is deteriorated, and when the concentration of the primer is 0.5. mu.M/L, the amplification efficiency is stable and non-specific amplification is not caused; the use amount of Pfu DNA polymerase has great influence on the amplification efficiency of PCR, and when 5.0U of Pfu DNA polymerase is added into each system, the amplification efficiency is higher and the most economical; the annealing temperature was selected to be 52 ℃ and the annealing time was 30s, the PCR amplification efficiency was high, and the results were stable, as shown in FIG. 1.
Example 2
This example studies the detection and analysis of some clinical problematic species, and the results are shown below:
2.1 detection and analysis results of Boardella holmeii (Bordetella holmeii, B. holmeii)
Extracting total DNA of the isolate as a template, amplifying by using an optimized PCR reaction system and conditions, performing bidirectional sequencing on a product (a sequencing diagram is shown in figure 2), and then performing BLAST comparison with a Genebank database, wherein the result shows that the 16S rRNA sequence of the isolate is similar to the 16S rRNA sequence of B.holmesii reported in the database, and the similarity reaches 99 percent (figure 3), so that the strain is identified as B.holmesi. The 16S rRNA gene sequence of the isolate is uploaded to NCBI GenBank with the access number of KT 828544.1. B.holmesi has not been reported and separated in China, so the strain is uploaded to China general microbiological culture Collection center (CGMCC, the preservation number of the strain is CGMCC 1.13721), and the certificate of strain preservation is shown in figure 4.
2.2 detection and analysis results of Brucella melitensis (B. melitensis)
Extracting the total DNA of the isolate as a template, sequencing the product after the 16S rRNA gene PCR amplification (the sequencing diagram is shown in figure 5), and performing sequence comparison by using online BLAST software, wherein the similarity with B.melitensis reaches 100 percent (figure 6), and the strain is identified as maltas brucella.
2.3 detection and analysis results of Campylobacter jejuni (C. jejuni)
Extracting the total DNA of the isolate as a template, sequencing the product after the PCR amplification of the 16S rRNA gene (the sequencing diagram is shown in figure 7), and performing sequence comparison by using online BLAST software, wherein the similarity with C.jejuni reaches 100 percent (figure 8), and the strain is identified as campylobacter jejuni.
The 16S rRNA sequence has both stability and high variability, and with the rapid development of the current molecular biology technology, the 16S rRNA gene sequence analysis has become a common method for the accurate identification of bacteria. By using 16S rRNA gene sequence analysis, the clinical microbiology laboratory can realize rapid, simple and accurate classification and identification of microorganisms, and can further research and analyze bacteria from the aspects of molecular level and genetic evolution. The inventor establishes a method for effectively identifying the difficult and complicated strains by applying a 16S rRNA gene sequence analysis technology according to laboratory conditions, expands the identification spectrum of clinical strains and meets the clinical identification requirement on the difficult and complicated strains.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Reference to the literature
[1]Drancourt M,Bollet C,Carlioz A,et al.16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates.J Clin Microbiol 2000；38(10):3623-3630.
[2]Woo PC,Lau SK,Teng JL,et al.Then and now:use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories.Clin Microbiol Infect 2008；14(10):908-934.
[3]Tang YW,Ellis NM,Hopkins MK,et al.Comparison of phenotypic and genotypic techniques for identification of unusual aerobic pathogenic gram-negative bacilli.J Clin Microbiol 1998；36(12):3674-3679.
[4]Rodicio Mdel R,Mendoza Mdel C.Identification of bacteria through 16S rRNA sequencing: principles,methods and applications in clinical microbiology.Enferm Infecc Microbiol Clin 2004；22(4):238-245.
[5]Martini M,Lee IM,Bottner KD,et al.Ribosomal protein gene-based phylogenyfor finer differentiation and classification of phytoplasmas.Int J Syst Evol Microbiol 2007；57(Pt 9):2037-2051.
[6]Tortoli E,Bartoloni A,
EC,et al.Burden of unidentifiable mycobacteria in a reference laboratory.J Clin Microbiol 2001；39(11):4058-4065.
[7]Shamputa IC,Rigouts And L,Portaels F.Molecular genetic methods for diagnosis and antibiotic resistance detection of mycobacteria from clinical specimens.APMIS 2004；112(11-12):728-752.
[8]Basein T,Gardiner BJ,Andujar Vazquez et al.Microbial Identification Using DNA Target Amplification and Sequencing:Clinical Utility and Impact on Patient Management.Open Forum Infect Dis 2018；5(11):ofy257.
[9]Kolbert CP,Persing DH.Ribosomal DNA sequencing as a tool for identification of bacterial pathogens.Curr Opin Microbiol 1999；2(3):299-305.
[10]Woo PC,Ng KH,Lau SK,et al.Usefulness of the MicroSeq 500 16S ribosomal DNA-based bacterial identification system for identification of clinically significant bacterial isolates with ambiguous biochemical profiles.J Clin Microbiol 2003；41(5):1996-2001.
[11]Woo PC,Chung LM,Teng JL,et al.In silico analysis of 16S ribosomal RNA gene sequencing-based methods for identification of medically important anaerobic bacteria.J Clin Pathol 2007；60(5):576-579.
[12]Cloud JL,Neal H,Rosenberry R,et al.Identification of Mycobacterium spp.by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries.J Clin Microbiol 2002；40(2):400-406.
[13]Clarridge JE 3rd.Impact of 16S rRNA gene sequence analysis for identificationof bacteria on clinical microbiology and infectious diseases.Clin Microbiol Rev 2004；17(4):840-862.
[14] Wen, Wanyarespectively, Pengxiang, et al, rapid detection of common pathogens using the Universal primer PCR in combination with SSCP and RFLP techniques, J.Zhonghua microbiology and immunology 2001; 21(6):687-689.
[15]Mignard S,Flandrois JP.16S rRNA sequencing in routine bacterial identification:a 30-month experiment.J Microbiol Methods 2006；67(3):574-581。
Claims (10)
1. A method for identifying clinical problematic strains is characterized in that a 16S rRNA gene sequence analysis method is adopted, and comprises the following steps:
s1: extracting bacterial DNA of a strain to be identified, selecting a bacterial universal primer, and establishing a PCR method for amplifying bacterial 16S rRNA;
s2: optimizing a PCR reaction system and reaction conditions to obtain an amplification product;
s3: and comparing the amplified product with a gene database to obtain the strain of the bacteria to be identified.
2. The method of claim 1, wherein the bacterial universal primer is a 27F and 1492R universal primer.
3. The method of claim 1, wherein the PCR reaction system comprises a primer, Pfu Buffer, dNTP, Pfu DNA polymerase, DNA template, and ddH2O。
4. The method of claim 3, wherein the primer concentration is 0.1-0.75 μ M/L; preferably, the primer concentration is 0.5. mu.M/L.
5. The method according to claim 3, wherein the Pfu DNA polymerase is used in an amount of 2.5 to 7.0U/system; preferably, the Pfu DNA polymerase is used in an amount of 5.0U/system.
6. The method of claim 1, wherein the PCR reaction conditions are: pre-denaturation at 95 ℃ for 5min, followed by 35 cycles of amplification (denaturation at 95 ℃ for 30s, annealing at 52-55 ℃ for 10-40s, extension at 72 ℃ for 90s), followed by extension at 72 ℃ for 7 min.
7. The method of claim 6, wherein the PCR annealing temperature is 52 ℃; preferably, the annealing time is 30 s.
8. The method according to claim 1, wherein at S3, the species of the bacteria to be identified is obtained by performing a BLAST sequence alignment using the Genebank database.
9. The method of claim 1, wherein the bacteria comprise bordetella holtzeri, brunella maltas, and campylobacter jejuni.
10. Use of the method according to claims 1-9 for identifying a microbial species in a sample.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154847A (en) * | 2020-01-15 | 2020-05-15 | 北京睿博兴科生物技术有限公司 | Rapid nucleic acid extraction sequencing identification method based on bacterial 16S rDNA sequence |
| CN112593015A (en) * | 2021-01-08 | 2021-04-02 | 中国人民解放军总医院第一医学中心 | Primer composition, sequencing kit and detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154847A (en) * | 2020-01-15 | 2020-05-15 | 北京睿博兴科生物技术有限公司 | Rapid nucleic acid extraction sequencing identification method based on bacterial 16S rDNA sequence |
| CN112593015A (en) * | 2021-01-08 | 2021-04-02 | 中国人民解放军总医院第一医学中心 | Primer composition, sequencing kit and detection method |
| CN114373508A (en) * | 2022-01-24 | 2022-04-19 | 浙江天科高新技术发展有限公司 | Strain identification method based on 16S rDNA sequence |
| CN114373508B (en) * | 2022-01-24 | 2024-02-02 | 浙江天科高新技术发展有限公司 | Strain identification method based on 16S rDNA sequence |
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