阅读说明：本技术 一种基于16S rRNA基因设计的巢式PCR检测细菌性微生物核酸DNA的方法 (Method for detecting nucleic acid DNA of bacterial microorganisms by nested PCR based on 16S rRNA gene design ) 是由 田国忠 于 2019-10-09 设计创作，主要内容包括：一种基于16S rRNA基因设计的巢式PCR检测细菌性微生物核酸DNA的方法,涉及分子生物学技术领域,涉及待检样品中细菌性病原体核酸DNA的巢式PCR检测技术,通过对巢式PCR扩增产物的测序,鉴定细菌性病原体种类。基于16S rRNA基因设计的巢式PCR检测细菌性微生物核酸DNA的方法,灵敏性高,适用范围广,适用所有的目前已命名的细菌性微生物的检测与鉴定,该发明操作简单,费用低。(A nested PCR detection method for bacterial microbial nucleic acid DNA based on 16S rRNA gene design relates to the technical field of molecular biology, relates to a nested PCR detection technology for bacterial pathogen nucleic acid DNA in a sample to be detected, and identifies the bacterial pathogen species through sequencing of nested PCR amplification products. The method for detecting the nucleic acid DNA of the bacterial microorganisms by nested PCR based on 16S rRNA gene design has high sensitivity and wide application range, is suitable for detecting and identifying all the currently named bacterial microorganisms, and has simple operation and low cost.)

1. A method for detecting and identifying nucleic acid DNA of bacterial microorganisms by nested PCR based on 16S rRNA gene design is characterized by comprising the following steps:

(1) extracting nucleic acid DNA of a sample to be detected;

(2) designing a primer pair: the nested PCR primers are designed by using software, and specific primer sequences comprise the following sequences:

first PCR amplification primer:

a forward primer: f1: 5 '-TAGCT/GGGTCTGAGAGGATGA-3', SEQ ID NO:1, wherein T/G is an ampholytic base;

the facultative base T/G is indicated by K, namely 5 '-TAGCKGGTCTGAGAGGATGA-3';

reverse primer: r1: 5'-ATGTGTGGCGGGCAGTGT-3', SEQ ID NO: 2;

second PCR amplification primer:

a forward primer: f2: 5'-TACGGGAGGCAGCAGT-3', SEQ ID NO: 3;

reverse primer: r2: 5'-ACTAGCGATTCCGACTTCA-3', SEQ ID NO: 4;

(3) taking the nucleic acid DNA extracted in the step 1 as a template, and performing first amplification on the nucleic acid DNA to be detected through PCR reaction by using primers shown in SEQ ID NO. 1-2 to obtain the nucleic acid DNA of a first PCR product; amplifying the nucleic acid DNA of the first PCR product for the second time through PCR reaction by using primers shown in SEQ ID NO. 3-4;

(4) detecting and analyzing the PCR amplification product;

(5) sequencing a PCR product;

(6) and (5) analyzing the nucleotide sequence sequenced in the step (5) to determine the type of the bacterial microorganism.

2. The method for detecting nucleic acid DNA of bacterial microorganisms by nested PCR based on 16S rRNA gene design according to claim 1, wherein the extraction of nucleic acid DNA of the sample to be detected: nucleic acid DNA extracted from samples to be tested, including milk, water and interstitial fluid.

3. The method for nested PCR based on 16S rRNA gene design for the detection of nucleic acid DNA of bacterial microorganisms according to claim 1, wherein the PCR amplification reaction: adding the corresponding nucleic acid DNA into the corresponding PCR reaction mixed solution for PCR amplification; the PCR reaction mixed solution comprises: PCR Buffer 10 x Buffer, 4 kinds of dNTP mixture solution, Taq enzyme, primer and double distilled water mixture.

4. The method for detecting nucleic acid DNA of bacterial microorganisms by nested PCR based on 16S rRNA gene design according to claim 2,

as a preferred embodiment of the invention, the nested PCR reaction system comprises a PCR Buffer solution 10 × Buffer (Mg)²⁺15mmol/L)2.5 muL, 2 muL of 4 kinds of dNTP mixture solution, 0.2 muL of Taq enzyme (TaKaRa LA Taq enzyme 5U/muL), 0.4 muL of each of forward primer F1 solution and reverse primer R1 solution (the concentration is 10 mumol/L), 4 muL of extract of nucleic acid DNA of a sample to be detected, and double distilled water is supplemented to 25 muL volume;

as a preferred embodiment of the invention, the nested PCR reaction system comprises a PCR Buffer solution 10 × Buffer (Mg)²⁺15mmol/L) 2.5. mu.L, 2. mu.L of 4 kinds of dNTP mixture solution, 0.2. mu.L of Taq enzyme (TaKaRa LA Taq enzyme 5U/. mu.L), 0.4. mu.L each of forward primer F2 solution and reverse primer R2 solution, 4. mu.L of first PCR amplification product nucleic acid DNA solution, and double distilled water was supplemented to a volume of 25. mu.L.

5. The method for detecting nucleic acid DNA of bacterial microorganisms by nested PCR based on 16S rRNA gene design according to claim 3, wherein the concentration of each primer solution is 10. mu. mol/L; the concentration of each NTP, A, T, G and C, in the dNTP mixture solution was 2.5 mmol/L.

6. The method for detecting nucleic acid DNA of bacterial microorganisms by nested PCR based on 16S rRNA gene design according to claim 1, wherein the nested PCR, the first PCR reaction, is performed under the following conditions: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 45sec, annealing at 52 ℃ for 45sec, extension at 72 ℃ for 90sec, 30 cycles; extending for 5min at 72 ℃; the conditions for the second PCR reaction were: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 45sec, annealing at 52 ℃ for 45sec, extension at 72 ℃ for 90sec, 30 cycles; extension at 72 ℃ for 5 min.

7. The method for detecting nucleic acid DNA of bacterial microorganisms by nested PCR based on 16S rRNA gene design according to claim 1, wherein the PCR amplification products are detected and analyzed in step (4); after the reaction is finished, carrying out 1% agarose gel electrophoresis; the 1% agarose gel is agarose: the mass-to-volume ratio of the gel is 0.01 g/ml; performing gel imaging analysis on the PCR product after electrophoresis for 1h by a 90V voltage and 1% agarose gel; if the detected sample has 1006bp band amplified by the second PCR, the different pathogens have different sizes and are bright and clear, which indicates the existence of bacterial microbe, and can be used for sequencing analysis.

8. The method for detecting nucleic acid DNA of bacterial microorganisms by nested PCR based on 16S rRNA gene design according to claim 1, wherein the PCR product sequencing of step (5): the sequencing primers are a forward primer F2 and a reverse primer R2, and the sequencing is performed in a bidirectional mode until the sequencing is completed.

9. The method for nested PCR detection of nucleic acid DNA of bacterial microorganisms based on 16S rRNA gene design according to claim 1, wherein the nucleotide sequence analysis in step (6) has two methods, optionally one;

the first method, which is to identify the type of the bacterial microorganism by subjecting the nucleotide sequence to NCBI, BLAST, and performing a matching analysis, and the highest match (Max score) as a result (sequencing differentiating matching searching algorithms);

the second method adopts MEGA5.1 software to compare and analyze with the nucleotide sequence of the 16S rRNA of the known bacterial microorganism, and the types of the bacterial microorganisms can be identified as the result of cluster analysis, and the nucleic acid DNA sequence of the sample to be detected is consistent with the known nucleic acid DNA sequence.

Technical Field

The invention relates to the technical field of molecular biology, in particular to a nested PCR (polymerase chain reaction) technical method for detecting and identifying the nucleic acid DNA of bacterial microorganisms in a sample to be detected (such as milk, water, interstitial fluid and the like), and the types of the bacterial microorganisms are identified by sequencing PCR amplification products.

Background

The sequence of the 16SrDNA comprises 10 variable regions (variable regions) and 11 constant regions (constant regions) which are alternated, the variable regions are different from bacteria to bacteria, and the variation degree is closely related to the phylogeny of the bacteria, so the 16SrDNA can be used as a phylogenetic marker molecule which is most commonly used for bacterial colony structure analysis. The characteristics of the 16S rDNA variable region and the constant region are utilized to classify and identify bacteria of different species.

PCR detection is to use polymerase chain reaction to amplify bacterial DNA fragments, sequence, and analyze nucleotide sequence, thereby qualitatively judging the type of bacteria. The method takes 16SrDNA as a target gene, and designs a primer which only amplifies the bacterial 16 SrDNA. It is not amplified to virus and human leucocyte DNA. With respect to the primers and probes commonly used for 16SrDNA, a slightly shorter target sequence works better when the extracted DNA may be damaged; when the bacteria content in the sample is rich, the long target sequence is more suitable, usually, the longer target sequence can provide more variable sequences, which is beneficial to more accurate identification, and in addition, different PCR primer pairs have different amplification efficiency. Therefore, before selecting a primer pair, the primers are tested and evaluated to determine whether they can be used for diagnosis,

the accuracy of reading the sequence is very high at present, the probability of an operation error is 0.01% -0.02%, the identification result of the bacteria is not influenced at the misreading level, and the overlapping region can be compared and corrected through positive and negative complementary sequence determination, so the probability of generating an error sequence is very low.

There is no general operating manual for identifying bacteria using 16S rDNA sequencing methods, and sequence analysis relies primarily on the percentage of sequence similarity. It is generally accepted that a similarity ratio > 99% is defined as species, > 95% and > 99% as genus, < 95% as family.

The invention designs common PCR, primer design selects nucleotide sequence with medium length, including constant region and variable region, in order to improve the sensitivity, selects nested PCR secondary amplification, the product is sequenced, the sequence is compared and analyzed, thereby identifying the species of bacterial microorganism.

Disclosure of Invention

The invention aims to provide a nested PCR detection method for quickly and specifically detecting and identifying the nucleic acid DNA of bacterial microorganisms, and the method is used for non-disease diagnosis.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for detecting nucleic acid DNA of a bacterial microorganism based on nested PCR designed on the basis of 16S rRNA gene comprises the following steps:

(1) extracting the nucleic acid DNA of a sample to be detected, wherein the nucleic acid DNA of the sample to be detected is extracted from milk, water and interstitial fluid waiting for detection of the sample;

(2) designing a primer pair: bacterial microorganism 16S rRNA nested PCR primers are respectively designed by application software such as Primer 5.0 software, and specific Primer sequences comprise the following:

first PCR amplification primer:

a forward primer: f1: 5 '-TAGCT/GGGTCTGAGAGGATGA-3', SEQ ID NO:1, wherein T/G is an ampholytic base; the facultative base T/G is indicated by K, namely 5 '-TAGCKGGTCTGAGAGGATGA-3';

reverse primer: r1: 5'-ATGTGTGGCGGGCAGTGT-3', SEQ ID NO: 2;

second PCR amplification primer:

a forward primer: f2: 5'-TACGGGAGGCAGCAGT-3', SEQ ID NO: 3;

reverse primer: r2: 5'-ACTAGCGATTCCGACTTCA-3', SEQ ID NO: 4;

(3) taking the nucleic acid DNA extracted in the step (1) as a template, and carrying out first amplification on the template nucleic acid DNA through PCR reaction by using primers shown in SEQ ID NO. 1-2 to obtain the nucleic acid DNA of a first PCR product; amplifying the nucleic acid DNA of the first PCR product for the second time through PCR reaction by using primers shown in SEQ ID NO. 3-4;

wherein the PCR amplification reaction: adding the corresponding nucleic acid DNA to be detected into the corresponding PCR reaction mixed solution for PCR amplification; the PCR reaction mixed solution comprises: a mixed solution of a sample nucleic acid DNA to be detected, a PCR Buffer solution of 10 multiplied by Buffer, 4 dNTP mixture solutions, Taq enzyme, a primer and double distilled water;

(4) detecting and analyzing the PCR amplification product; after the reaction is finished, carrying out 1% agarose gel electrophoresis; the 1% agarose gel is agarose: the mass-to-volume ratio of the gel is 0.01 g/ml; the PCR product was subjected to gel imaging analysis after electrophoresis for 1 hour on a 1% agarose gel at 90V.

If the detected sample has a band with the size of 1006bp (different pathogens have different sizes) amplified by the second PCR of the electrophoresis result, the detected sample is bright and clear, and bacterial microorganisms are suggested to exist, so that the detected sample can be used for sequencing analysis; if the electrophoresis result shows that the detected sample has no band in the second PCR amplification, the detected sample has no bacterial microorganism nucleic acid DNA or the amount of the nucleic acid DNA is as small as that which can not be detected by PCR;

(5) sequencing a PCR product: sequencing primers are a forward primer F2 and a reverse primer R2, and bidirectional sequencing is carried out until the sequencing is completed;

(6) analyzing the nucleotide sequence sequenced in the step (5) to determine the type of the bacterial microorganism; there are two methods for nucleotide sequence analysis, one of which is optional;

the first method, the nucleotide sequence is submitted to NCBI, BLAST for matching analysis, and the result (sequence partitioning matching highest identifiers) (Max score) is the highest match, namely, the pathogen of infection can be determined;

the second method adopts MEGA5.1 software to carry out comparison analysis with the nucleotide sequence of the 16S rRNA of the known bacterial microorganism, and the result of cluster analysis shows that the nucleic acid DNA sequence of the sample to be detected is consistent with the known nucleic acid DNA sequence, thus the type of the bacterial microorganism can be determined;

as a further preferable scheme of the invention: the nested PCR reaction comprises two times of PCR, a first time of PCR and a second time of PCR;

as a further preferable scheme of the invention: the template nucleic acid DNA in the first PCR system is the nucleic acid DNA extracted from the sample to be detected;

as a preferred embodiment of the present invention: the template nucleic acid DNA in the second PCR system is the nucleic acid DNA of the first PCR reaction product;

as a preferred embodiment of the present invention: the nested PCR reaction system comprises:0.4. mu.L of each of the forward primer F1 solution and the reverse primer R1 solution, and 10 × Buffer (Mg)²⁺15mmol/L)2.5 muL, 2 muL of 4 kinds of dNTP mixture solution, 0.2 muL of Taq enzyme (TaKaRa LA Taq enzyme 5U/muL), 4 muL of extracting solution of nucleic acid DNA of a sample to be detected, and supplementing double distilled water to 25 muL volume;

as a preferred embodiment of the invention, the nested PCR reaction system comprises a PCR Buffer solution 10 × Buffer (Mg)²⁺15mmol/L) 2.5. mu.L, 2. mu.L of 4 kinds of dNTP mixture solution, 0.4. mu.L each of forward primer F2 solution and reverse primer R2 solution, 0.2. mu.L of Taq enzyme (TaKaRa LA Taq enzyme 5U/. mu.L), 4. mu.L of first PCR amplification product nucleic acid DNA solution, and double distilled water to a volume of 25. mu.L;

as a further preferable scheme of the invention: the concentration of each primer solution was 10. mu. mol/L.

As a further preferable scheme of the invention: the concentration of each NTP (A, T, G and C) in the dNTP mixture solution is 2.5 mmol/L.

As a further preferable scheme of the invention: in the first PCR reaction system, a nucleic acid DNA template to be detected is added as the nucleic acid DNA extracted from the tissue specimen, and the adding amount is 4 mu L. In the second PCR reaction system, the amount of the nucleic acid DNA template to be detected is the first PCR amplification product, and the adding amount is 4 mu L.

As a further preferable scheme of the invention: the nested PCR is carried out under the conditions of the first PCR reaction: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 45sec, annealing at 52 ℃ for 45sec, extension at 72 ℃ for 90sec, 30 cycles; extension at 72 ℃ for 5 min.

As a further preferable scheme of the invention: the conditions of the nested PCR and the second PCR reaction are as follows: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 45sec, annealing at 52 ℃ for 45sec, extension at 72 ℃ for 90sec, 30 cycles; extension at 72 ℃ for 5 min.

Compared with the prior art, the invention has the beneficial effects that: the single 16S rRNA primer used at present is difficult to reach the level of detecting trace nucleic acid DNA, and the single pathogen specific primer can not reach the purpose of detection, the nested PCR technology of the invention has high sensitivity, can detect trace nucleic acid DNA, has wide application range, and can identify the type of the known named bacterial microorganism; the invention has simple operation, can be completed by personnel with common PCR technology, and can reach the actual requirements on sequencing price and timeliness.

Drawings

FIG. 1 is an electrophoresis diagram of an example of nested PCR based on 16S rRNA gene design for detecting bacterial microorganisms in a cerebrospinal fluid specimen;

the forward primer F1 and the reverse primer R1 are used for carrying out the first PCR amplification on sample nucleic acid DNA, the amount of the sample nucleic acid DNA added into a PCR reaction system is 4 mu L, the product of the sample nucleic acid DNA is subjected to the second PCR amplification by the forward primer F2 and the reverse primer R2, and the amount of the first PCR product nucleic acid DNA added into the PCR reaction system is 4 mu L, so that a clear and bright electrophoresis band is formed at the 1006bp position. The second PCR product was sent to the sequencing company for sequencing. 96 clinically collected cerebrospinal fluid samples are detected in total, and 21 bacterial microorganisms exist through nested PCR detection.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments, and are not to be taken as limitations of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.