Bacteria 16s rDNA full-length RAA detection kit and method for identifying bacteria by using bacterial 16s rDNA full-length RAA detection kit

Abstract

The invention belongs to the technical field of biological detection, and relates to a bacterium 16s rDNA full-length RAA detection kit and a method for identifying bacteria by using the same. The kit comprises a primer pair for RAA amplification of bacterium 16srDNA full length, the sequence of an upstream primer of the primer pair is as shown in SEQ ID NO: 1, and the sequence of a downstream primer of the primer pair is as shown in SEQ ID NO: 2. According to the bacteria 16s rDNA full-length RAA detection kit and the method for identifying the bacteria by using the same, high-specificity, high-sensitivity and high-stability RAA amplification can be performed on the 16s rDNA full lengths of various bacteria in a sample to be detected, and amplification products are easier to quickly and accurately determine the species of the bacteria in the sample to be detected through sequencing and sequence comparison.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and relates to a bacterial 16s rDNA full-length RAA detection kit and a method for identifying bacteria using the same. Background Art

[0002] Bacteria contain three types of rDNA sequences: 23S, 16S, and 5S. 16S rDNA is the most commonly used molecular clock in bacterial systematics due to its moderate number of nucleotides, high information content, high stability, and ease of extraction and analysis. 16S rDNA encodes the small subunit rRNA (rRNA) of the prokaryotic ribosome. It is approximately 1540 base pairs long and is present in the chromosomal genome of all bacteria. The 16S rRNA molecule contains nine variable and ten constant regions. Its moderate size of approximately 1.5 kilobases (kb) allows it to capture the diversity of bacterial genera while being relatively easy to sequence using sequencing technologies, making it a popular choice among bacteriologists and taxonomists.

[0003] 16S rDNA identification refers to the use of bacterial 16S rDNA sequence sequencing to identify bacterial species. It is a method for quickly obtaining bacterial species information, which includes steps such as bacterial genomic DNA extraction, 16S rDNA specific primer amplification, amplification product purification, DNA sequencing, and sequence alignment.

[0004] In 16S rRNA molecules, the variable region sequence varies among bacteria, while the constant region sequence is largely conserved. Therefore, primers can be designed using the constant region sequence to amplify 16S rDNA fragments, and differences in the variable region sequence can be used to classify and identify bacteria of different genera and species. The first 500bp of the 16S rDNA sequence varies significantly and contains a wealth of species-specific information. Therefore, for most strains, only the first pair of primers needs to sequence the first 500bp to identify the bacterial genus. For publication in scientific papers or when the first 500bp cannot be identified, full-length 16S rDNA amplification and sequencing is necessary to obtain more comprehensive 16S rDNA sequence information.

[0005] Currently, the United States Pharmacopoeia (USP 40 <1113> ), European Pharmacopoeia (EP 9.0<5.1.6>), Japanese Pharmacopoeia (JP17<General Information G4> ) and the Chinese Pharmacopoeia (General Chapter 9204) both include chapters on the use of molecular biology techniques for drug microbial quality control. Microbial identification methods based on 16S rRNA nucleic acid sequencing technology have been established and applied to the study of clinical isolates, but their use in drug quality control and the identification of common contaminating bacteria in drug production environments has rarely been reported. In literature on microbial identification using 16S rRNA nucleic acid sequencing, the selected sequencing targets and sequencing primers vary, and the DNA characteristic sequence fragments with identification and classification significance that meet drug quality control requirements are not yet clear. Most of the nucleic acid sequencing results in existing studies are compared with nucleic acid sequences in the Genebank database using the Blast analysis method. There is little attention paid to the quality verification of sequencing results and the accuracy of sequence information in the Genebank database, and the basis for judging sequencing results is not standardized and unified.

[0006] In addition, there are many commercial bacterial 16S rDNA amplification and capture kits on the market, but they all use PCR for amplification and capture. Some literature also reports on the use of alkaline lysis method for extracting bacterial genomes. Some laboratories widely use the water boiling method to crudely extract bacterial genomes for subsequent 16S rDNA PCR amplification and sequencing for bacterial species identification. The details are as follows:

[0007] 1) Conventional PCR amplification of bacterial 16S rRNA usually takes 2-3 hours, which is a long time;

[0008] 2) Some alkaline lysis methods for extracting and amplifying bacterial genomic nucleic acids reported in the literature are not simple enough and have limited applicability.

[0009] 3) Conventional boiling methods are difficult to effectively destroy the cell walls of Gram-positive bacteria, resulting in extremely low efficiency in genomic DNA extraction and making it unsuitable for nucleic acid sequencing. Summary of the Invention

[0010] The primary purpose of the present invention is to provide a bacterial 16s rDNA full-length RAA detection kit that can perform high-specificity, high-sensitivity, and high-stability RAA amplification on the full-length 16s rDNA of various bacteria in the sample to be detected, and make the amplified products easier to sequence and compare, so as to quickly and accurately determine the species of the bacteria in the sample to be detected.

[0011] To achieve this object, in a basic embodiment, the present invention provides a bacterial 16srDNA full-length RAA detection kit, which comprises a primer pair for RAA amplification of the full-length bacterial 16srDNA, the sequence of the upstream primer of the primer pair is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2.

[0012] In a preferred embodiment, the present invention provides a bacterial 16s rDNA full-length RAA detection kit, wherein the kit further comprises dNTPs, dithiothreitol, a ribonuclease inhibitor, a recombinase T4 UvsX, a single-strand binding protein gp32, a T4 UvsY auxiliary protein, a strand-displacing Bsu DNA polymerase, creatine kinase, and a magnesium acetate solution.

[0013] The recombinase T4 UvsX is a recombinase with pairing and strand transfer activities. The single-stranded binding protein gp32 can stabilize the transiently formed ssDNA region. The T4 UvsY auxiliary protein can promote the binding of the recombinase to single-stranded DNA. The strand-displacing Bsu DNA polymerase is a DNA isothermal amplification polymerase with strand-displacing activity. Creatine kinase can stimulate the decomposition of ATP and creatine into phosphocreatine and ADP and release energy.

[0014] The second object of the present invention is to provide a method for identifying bacteria, which can use the sequencing results of bacterial 16s rDNA obtained by RAA amplification of the sample to be tested to quickly and accurately determine the species of bacteria in the sample to be tested by comparing them with the bacterial 16s rDNA standard sequence.

[0015] To achieve this object, in a basic embodiment, the present invention provides a method for identifying bacteria, wherein the method comprises the following steps:

[0016] (1) Extracting bacterial DNA from the sample to be tested;

[0017] (2) performing RAA amplification on the bacterial DNA extracted in step (1) using the kit described above to obtain an amplified product;

[0018] (3) constructing a sequencing library and sequencing the amplified products;

[0019] (4) Compare the sequencing results with the 16s rDNA standard sequences of various bacteria to determine the species of bacteria in the sample to be tested.

[0020] In a preferred embodiment, the present invention provides a method for identifying bacteria, wherein in step (1), bacterial DNA of a sample to be detected is extracted using a bacterial DNA extraction kit.

[0021] In a preferred embodiment, the present invention provides a method for identifying bacteria, wherein in step (2), the concentration of the primers during RAA amplification is 5-20 μmol / L.

[0022] In a preferred embodiment, the present invention provides a method for identifying bacteria, wherein in step (2), RAA amplification is performed at 39-42° C. for 15-25 minutes.

[0023] In a preferred embodiment, the present invention provides a method for identifying bacteria, wherein in step (2), the obtained amplification product is further purified using magnetic beads.

[0024] In a preferred embodiment, the present invention provides a method for identifying bacteria, wherein in step (3), the sequencing library is a third-generation sequencing library.

[0025] In a preferred embodiment, the present invention provides a method for identifying bacteria, wherein in step (3), the third-generation sequencing library is constructed using Nanopore's ligation sequencing kit and Native tagging kit.

[0026] In a preferred embodiment, the present invention provides a method for identifying bacteria, wherein in step (3), the constructed sequencing library is also subjected to quality control.

[0027] In a preferred embodiment, the present invention provides a method for identifying bacteria, wherein the bacteria include Gram-negative bacteria and Gram-positive bacteria.

[0028] The beneficial effects of the present invention are that the bacterial 16s rDNA full-length RAA detection kit and the method for identifying bacteria using the same can perform RAA amplification of the full-length 16s rDNA of various bacteria in the sample to be detected with high specificity, high sensitivity and high stability, and make the amplified products easier to determine the species of the bacteria in the sample to be detected quickly and accurately through sequencing and sequence comparison.

[0029] By comparing the full-length bacterial 16s rDNA sequences obtained from the NCBI database, the present invention selected highly conserved segments and designed primer pairs capable of covering all bacterial 16s rDNA sequences. By adjusting and optimizing the primer pair sequences and amplification conditions, such as primer concentration and reaction conditions, the present invention optimizes the amplification efficiency of the primer pair, enabling efficient RAA (recombinase-aided amplification) amplification reaction, shortening the capture time of the current PCR method by 83.3%. By constructing libraries for the RAA amplification products according to the third-generation sequencing library construction protocol, full-length bacterial 16s rDNA sequencing can be achieved on different platforms.

[0030] The kit provided by the present invention performs capture and library construction sequencing on different bacteria, and can achieve 100% detection of bacterial species. It is comparable to the common PCR capture sequencing method but shortens the time by 83.3%, greatly improving the sequencing efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The capillary electrophoresis patterns of the products obtained after RAA amplification of bacterial DNA from 8 incoming ship ballast water samples, 5 bacterial standard strain DNAs, and 1 fungal standard strain DNA are shown. A1 (L) represents the marker, B1 to H1 and A2 represent ballast water samples from incoming ships 1# to 8#, respectively, B2 represents the standard strain of Aspergillus flavus (CGMCC 3.15726), C2 represents the standard strain of Vibrio parahaemolyticus (CGMCC 1.1997), D2 represents the standard strain of Vibrio cholerae (CGMCC 1.8676), E2 represents the standard strain of Salmonella typhimurium (CGMCC 1.1190), F2 represents the standard strain of Shigella dysenteriae (CGMCC 1.1869), G2 represents the standard strain of Staphylococcus aureus (CGMCC 1.10755), and H2 represents the negative control.

[0032] Figure 2 The bacterial species composition of 8 ballast water samples from incoming ships in the third-generation sequencing is shown. DETAILED DESCRIPTION

[0033] In order to better understand the technical solutions and advantages of the present invention, the present invention is further described below through embodiments in conjunction with the accompanying drawings.

[0034] Example 1: Extraction of bacterial nucleic acid from samples

[0035] (1) Sample source

[0036] Eight ballast water samples from ships entering Ningbo Port, five bacterial standard strains and one fungal standard strain were used.

[0037] (2) Sample pretreatment

[0038] A 500 ml volume of ballast water sample from incoming ships was enriched and concentrated using a 0.5 μm filter membrane, and then eluted with 1 ml of sterile DEPC water for subsequent nucleic acid extraction.

[0039] The standard strains were enriched with 5 ml of the corresponding enrichment solution for 18 to 24 hours, and then 1 ml of the enrichment solution was centrifuged at 12,000 rpm for 2 minutes. After removing the supernatant, the bacteria were resuspended in 200 μl of sterile DEPC water for subsequent nucleic acid extraction.

[0040] (3) Extraction of bacterial nucleic acid

[0041] Bacterial nucleic acids were extracted from the samples using the QIAamp DNA Mini Kit according to the manufacturer's instructions. The specific steps are as follows:

[0042] Add 20 μl of proteinase K to 200 μl of sample, vortex for 15 seconds, mix thoroughly, and incubate at room temperature for 10 minutes. After incubation, centrifuge briefly, add 200 μl of lysis buffer to the sample, vortex for 15 seconds, mix thoroughly, and incubate at room temperature for 10 minutes. Then, add 200 μl of anhydrous ethanol to the resulting sample solution, vortex for 15 seconds, and centrifuge briefly. Pipette 620 μl of sample solution into the column placed in a 2 ml collection tube, centrifuge at 8000 rpm for 1 minute, place the column in a new 2 ml collection tube, and discard the old tube. Repeat the previous step to filter the remaining sample solution. Add 500 μl of AW1 solution to the column, centrifuge at 8000 rpm for 1 minute, place the column in a new 2 ml collection tube, and discard the old tube. Add 500 μl of AW2 solution to the column and centrifuge at 13000 rpm for 3 minutes. After discarding the liquid in the collection tube, centrifuge the column at 13,000 rpm for 1 minute. Place the column in a new 1.5 ml centrifuge tube and discard the old tube. Add 40 μl of AVE solution to the column and incubate at room temperature for 1 minute. Centrifuge at 8,000 rpm for 1 minute, discard the column, and obtain the viral nucleic acid. Store at -80°C until further use. Alternatively, repeat the extraction step without discarding the column; this process can increase DNA recovery by 20%.

[0043] Example 2: RAA amplification primer design

[0044] The full-length bacterial 16S rDNA sequence was obtained using the public database NCBI, and conserved segments were identified through bioinformatics analysis. Similar to conventional PCR primer design methods, highly conserved segments of the bacterial 16S rDNA were screened and a primer pair was designed to cover all bacterial species within this segment. This primer pair consists of two primers, an upstream primer and a downstream primer:

[0045] Upstream primer: GTGATCCNACCGCYGGMTCCCCTAC (SEQ ID NO: 1)

[0046] Downstream primer: AGAGAGCYTGCTNTCGYGTGACGAGCG (SEQ ID NO: 2)

[0047] The N in the primer sequence represents A, C, G or T.

[0048] Example 3: RAA amplification of full-length bacterial 16S rDNA

[0049] The RAA amplification kit containing the RAA amplification reagent was used to perform RAA isothermal amplification on the bacterial nucleic acids extracted from the 8 ballast water samples of the incoming ships in Example 1, the nucleic acids of 5 bacterial standard strains, and the nucleic acid of 1 fungal standard strain to obtain RAA amplification products corresponding to the 14 samples. The specific steps are as follows:

[0050] Place the nucleic acid from each sample in a PCR reaction tube to obtain a nucleic acid solution. Take 10 μl of the nucleic acid solution to each tube and add 1 μl of 10 mM dNTPs, 1 μl of 100 mM DTT (dithiothreitol), 1 μl of ribonuclease inhibitor, 1 μl of recombinase T4 UvsX, 1.5 μl of single-strand binding protein gp32, 0.5 μl of T4 UvsY auxiliary protein, 2 μl of strand-displacing Bsu DNA polymerase, 0.5 μl of creatine kinase, 1.5 μl of magnesium acetate solution, and 22 μl of Nuclease-Free Water. After the addition is complete, gently flick to mix and centrifuge briefly. Then, dilute the primer pairs in Example 2 to 10 μM. Add 4 μl of the upstream primer and downstream primer to each of the two reaction tubes, mix thoroughly, and centrifuge. Then, perform the RAA amplification reaction. The reaction procedure is: 39°C, 20 min. After the reaction is completed, the RAA amplification reaction product is obtained. Each RAA amplification reaction product was purified using 1.5 volumes of Beckman AMpure XP magnetic beads, then washed with 80% ethanol and eluted with 15 μl of nuclease-free water to obtain each purified RAA product, which was used as a sample to be tested.

[0051] The purified RAA products of 8 ballast water samples from inbound ships, 5 bacterial standard strains of nucleic acid and 1 fungal standard strain of nucleic acid were subjected to capillary electrophoresis. The results are shown in Figure 1 .like Figure 1 As shown, 8 ballast water samples from incoming ships (corresponding to B1 to H1 and A2 in the figure) had clear corresponding bands, and the fragment size was approximately 1450 bp, which was consistent with the expected size; the standard strain of Aspergillus flavus (CGMCC 3.15726, corresponding to B2 in the figure) also had a clear corresponding band, but the fragment size was approximately 600 bp, which was inconsistent with the expected size; 5 standard bacterial strains (corresponding to C2 to G2 in the figure) had clear corresponding bands, and the fragment size was approximately 1450 bp, which was consistent with the expected size.

[0052] Example 4: Full-length third-generation sequencing of 16S rDNA of bacteria carried in ballast water samples of incoming ships and identification of bacteria contained in the samples

[0053] Purified RAA products were quantified using a Qubit 2.0 Fluorometer and the Qubit dsDNA BR Assay kit (ThermoFisher Scientific). Library construction was performed using the Ligation Sequencing kit (SQK-LSK110) and Native Barcoding Expansion 1-12 (EXP-NBD104). Based on the length of the amplified fragments, the optimal DNA input was 200 ng. DNA products or libraries were quantified using a Qubit 2.0 Fluorometer and the Qubit dsDNA HSA Assay kit. The recommended library loading amount is 100-300 ng.

[0054] The specific steps include: constructing a library according to the manufacturer's instructions, performing a quality control test, and obtaining a qualified library. The resulting library is injected into an R9.4.1 chip, which is then placed in a MinION MK1C for sequencing. Data analysis can be performed using analysis software from Hangzhou Boyi Technology Co., Ltd. or other bioanalysis software. Figure 2 The bacterial species composition of 8 ballast water samples from incoming ships in the third-generation sequencing is shown.

[0055] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these changes and variations. The above embodiments or implementation methods are merely illustrative of the present invention, and the present invention may also be implemented in other specific ways or other specific forms without departing from the gist or essential features of the present invention. Therefore, the described implementation methods should be regarded as illustrative and not restrictive in any respect. The scope of the present invention should be described by the appended claims, and any changes that are equivalent to the intent and scope of the claims should also be included within the scope of the present invention.

Claims

1. A bacterial 16s rDNA full-length RAA detection kit, characterized by: The kit comprises a primer pair for RAA amplification of the full length of bacterial 16s rDNA, the sequence of the upstream primer of the primer pair is shown as SEQ ID NO: 1, and the sequence of the downstream primer is shown as SEQ ID NO:

2.

2. The kit according to claim 1, wherein: The kit further comprises dNTP, dithiothreitol, ribonuclease inhibitor, recombinase T4 UvsX, single-strand binding protein gp32, T4 UvsY auxiliary protein, strand displacement Bsu DNA polymerase, creatine kinase, and magnesium acetate solution.

3. A method for identifying bacteria, characterized in that: The method comprises the following steps: (1) Extracting bacterial DNA from the sample to be tested; (2) performing RAA amplification on the bacterial DNA extracted in step (1) using the kit according to claim 1 or 2 to obtain an amplified product; (3) constructing a sequencing library and sequencing the amplified products; (4) Compare the sequencing results with the 16s rDNA standard sequences of various bacteria to determine the species of bacteria in the sample to be tested.

4. The method according to claim 3, wherein: In step (1), bacterial DNA of the sample to be tested is extracted using a bacterial DNA extraction kit.

5. The method according to claim 3, wherein: In step (2), the concentration of the primers during RAA amplification is 5-20 μmol / L.

6. The method according to claim 3, wherein: In step (2), RAA amplification is performed at 39-42°C for 15-25 minutes.

7. The method according to claim 3, wherein: In step (2), the obtained amplified product is also purified by magnetic beads.

8. The method according to claim 3, wherein: In step (3), the sequencing library is a third-generation sequencing library.

9. The method according to claim 8, characterized in that: In step (3), the third-generation sequencing library is constructed using Nanopore's ligation sequencing kit and Native labeling kit.

10. The method according to claim 3, wherein: In step (3), the constructed sequencing library is also quality checked.

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