A specific molecular target, primer, kit and method for detecting green balloon bacteria

Abstract

The present invention relates to a specific molecular target, primer, kit and method for detecting green aerobic bacteria, and belongs to the field of PCR detection technology. The nucleotide sequence of the specific molecular target is shown in SEQ ID NO: 1. The present invention also provides a primer for detecting a specific molecular target of green aerobic bacteria, the nucleotide sequence of the forward primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 3. The primers of the present invention are used to perform PCR amplification on the sample to be tested, and whether the amplified product contains the target band is detected to determine whether the sample contains green aerobic bacteria. The method has the advantages of high sensitivity and strong specificity, and can meet the needs of rapid and specific detection of duck-derived green aerobic bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of PCR detection, and specifically relates to a specific molecular target, primers, a kit and a method for detecting green balloon bacteria. Background Art

[0002] Green balloon fungus is a zoonotic conditional pathogen. When the body's immunity is low or the immune function is not sound, it can cause sepsis, bacteremia, endocarditis, urinary tract infection, meningitis and arthritis in humans. However, with the development of technology, the pathogen has been found in a variety of economic animals and farmed animals, such as pigs, sheep, cattle, mice, tilapia, shrimps and crabs. Existing studies have shown that green balloon fungus is seriously harmful to young animals, and also has certain harm to adult livestock and aquatic products. Pig arthritis and bovine mastitis caused by green balloon fungus have caused huge economic losses in animal husbandry and breeding.

[0003] At present, conventional bacterial isolation and culture, morphological observation, and physiological and biochemical testing methods are time-consuming and cumbersome, and are prone to contamination. The test results take about a week. With the development of molecular biology, the PCR method has become an effective means of rapid detection of pathogens. The genes of the tested bacteria are qualitatively analyzed to determine whether the target bacteria exist. At present, this technology has been widely used in animal husbandry, and can more effectively and quickly identify pathogens, providing great help for clinical diagnosis. However, according to domestic and foreign literature reports, the existing technology has not yet established a specific PCR detection method for green balloon bacteria. Summary of the invention

[0004] The purpose of the present invention is to provide a specific molecular target for detecting green balloon fungi, use the primers designed with the specific molecular target to perform PCR amplification on the DNA of the sample to be tested, and then detect whether the amplified product contains a specific band to determine whether the sample contains green balloon fungi, thereby achieving specific identification of green balloon fungi.

[0005] The second object of the present invention is to provide a primer for detecting a specific molecular target of green balloon bacteria.

[0006] The third object of the present invention is to provide a kit for detecting green balloon bacteria.

[0007] The fourth object of the present invention is to provide a method for detecting green balloon fungi.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A specific molecular target for detecting green balloon bacteria, wherein the nucleotide sequence of the specific molecular target is shown in SEQ ID NO: 1.

[0010] The present invention aims to identify a template sequence with PCR amplification specificity in the bacterial genomic DNA sequence, intercepts the kemr sequence, uses bwa to align to CARD, VAFD and 8 strains of green balloon bacteria, and uses samtools to filter out the sequences aligned to CARD and VAFD and the sequences not aligned to green balloon bacteria, aligns all KMER sequences to the NCBI NT database through BLAST, counts and analyzes the alignment results, screens the specific sequence of green balloon bacteria, and uses the screened specific sequence as a template sequence for PCR identification. BLAST primer is used to perform online alignment of primers, which has good accuracy and specificity, no non-specific amplification of other bacteria, and realizes PCR identification of green balloon bacteria.

[0011] The technical solution adopted by the primers for detecting the specific molecular target of green balloon bacteria of the present invention is:

[0012] A primer for detecting a specific molecular target of green balloon fungus, wherein the nucleotide sequence of the forward primer AU-F is shown in SEQ ID NO: 2, and the nucleotide sequence of the reverse primer AU-R is shown in SEQ ID NO: 3.

[0013] The primers of the present invention are obtained through whole genome comparative analysis. Specifically, the specific sequence of the green balloon fungus screened above is used as the template sequence for PCR identification, and the BLAST primer tool is used to design specific PCR primers. The template sequence amplified by the designed primers is 312 bp.

[0014] The primers for detecting the specific molecular target of green balloon fungus of the present invention have good accuracy and specificity, will not perform non-specific amplification on other bacteria, and can achieve specific identification of green balloon fungus.

[0015] The technical solution adopted by the kit for detecting green balloon fungus of the present invention is:

[0016] A kit for detecting green balloon fungus comprises the forward primer AU-F and the reverse primer AU-R.

[0017] The kit for detecting green balloon fungi of the present invention has the advantages of high sensitivity, strong specificity, etc., and can meet the needs of quickly and accurately detecting green balloon fungi.

[0018] Furthermore, the kit also includes ddH2O and 2×Es Taq Master Mix.

[0019] The technical solution adopted by the method for detecting green balloon bacteria of the present invention is:

[0020] A method for detecting green balloon fungi comprises the following steps: extracting DNA from a sample to be tested, amplifying the DNA with the primers to obtain an amplified product, and performing electrophoresis analysis on the amplified product to determine whether a specific band of green balloon fungi exists; if the specific band exists, it indicates that the sample to be tested contains green balloon fungi, otherwise, it indicates that the sample to be tested does not contain green balloon fungi.

[0021] The present invention obtains a specific gene that can be used to identify green balloon fungi through bioinformatics means, designs primers for the gene, and establishes a detection method. The method for detecting green balloon fungi of the present invention has the advantages of high sensitivity and strong specificity. Compared with the traditional green balloon fungi identification method, the cost is relatively lower, the detection speed is faster, the operation is simple, and it is not only easy to master, but also easy to promote, and can be implemented in general molecular laboratories.

[0022] Furthermore, the amplification reaction system includes 7 μL of ddH2O, 10 μL of 2×Es Taq Master Mix, 0.5 μL of the forward primer, 0.5 μL of the reverse primer and 2 μL of template DNA; the concentrations of the forward primer and the reverse primer are both 10 μmol / L.

[0023] Furthermore, the reaction conditions of the amplification are: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 30 seconds, annealing at 50-60°C for 30 seconds, extension at 72°C for 30 seconds, denaturation, annealing, and extension repeated for 30 cycles; final extension at 72°C for 10 minutes. Preferably, the annealing temperature is 57°C. The present invention optimizes the annealing temperature and finally determines that 57°C is the optimal annealing temperature.

[0024] Furthermore, the green balloon fungus is sourced from animal husbandry animals.

[0025] Furthermore, the livestock animal is one of duck, pig, chicken, sheep, mouse, cattle, shrimp and crab.

[0026] Furthermore, the amplified product was stored at 4°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of electrophoresis results in an experimental example of the present invention, wherein M is DL2000 DNA Marker, 1-3 are duck Murrayella, 4-6 are Staphylococcus aureus, 7-9 are Escherichia coli, 10-12 are Salmonella, 13-15 are Streptococcus, 16-17 are Pasteurella, 18-21 are sheep-derived green balloon bacteria, duck-derived green balloon bacteria, chicken-derived green balloon bacteria, and pig-derived green balloon bacteria, respectively;

[0028] Figure 2Schematic diagram of sensitivity detection results in the experimental example of the present invention, where M: DL2000 DNA Marker, 1: 1×10 4 CFU / mL, 2: 1×10 5 CFU / mL, 3: 1×10 6 CFU / mL, 4: 1×10 7 CFU / mL, 5: 1×10 8 CFU / mL, 6: 1×10 1 CFU / mL, 7: 1×10 2 CFU / mL, 8: 1×10 3 CFU / mL;

[0029] Figure 3 This is a preferred test for the annealing temperature in the present invention. DETAILED DESCRIPTION

[0030] The principle of screening the specific sequence of green balloon bacteria species in the present invention is as follows:

[0031] The assembled green balloon fungus genome sequence starts from the first base and moves every 20 bases to intercept a fixed length sequence of 300bp, which is called a Kmer sequence. If there is a continuous kemr sequence or a single kmer sequence that only exists in the green balloon fungus genome but not in the genomes of other species, it means that the sequence is a specific sequence of green balloon fungus. The specific sequence is used as a template sequence for PCR specific amplification and suitable primers are designed, and it is particularly noted that the designed primers are not for amplifying the full-length sequence but for a partial sequence of the template sequence.

[0032] The specific implementation method is to use bwa (genome sequence alignment tool) to align all kmer sequences to CARD, VAFD and 8 strains of green balloon fungi; the genome data of the 8 strains of green balloon fungi are from the green balloon fungi genome published in the ncbi genome database, and the strain numbers are CCUG4311, NCTC8251, ATCC 11563, FDAARGOS_249, UMB0240, LL1, NCTC7595, 32-41-5, and the corresponding genome numbers are GCA_001543285.1, GCA_900445095.1, GCA_000178435.1, GCA_002083135.2, GCA_002871935.1, GCA_000262085.1, GCA_900445105.1, GCA_002280005.1; CARD is a database that integrates drug resistance genes in various microorganisms and collects drug resistance gene sequences of various microorganisms; Virulence factor (Virulence factor Virulence factor (VFs) refers to molecules with virulence properties such as invasiveness and toxins produced by the metabolism of bacteria, viruses, fungi, etc. The pathogen virulence factor database VFDB was developed by the Chinese Academy of Medical Sciences. It collects and organizes the composition, structure, function, pathogenicity, virulence island, sequence and genome information of known virulence factors of more than 100 important medical pathogens from 24 genera; drug-resistant genes and virulence factors usually appear in a variety of bacteria, so the kmer sequences from drug-resistant genes and virulence factors should be screened out. The present invention uses the samtools tool to filter out the sequences that are aligned to CARD and VAFD; at the same time, the sequences that are not aligned to green balloon bacteria are filtered out, and all the remaining KMER sequences are aligned to the NCBI NT database through BLAST, and the alignment results are counted and analyzed to screen out specific nucleotide sequences that can only be aligned to green balloon bacteria.

[0033] BLAST primer was used for online comparison of primers, which had good accuracy and specificity, and no nonspecific amplification of other bacteria, thus achieving PCR identification of green balloon bacteria.

[0034] The present invention is further described below with reference to the embodiments.

[0035] 1. Example of detecting specific molecular targets of green balloon bacteria

[0036] Example 1

[0037] The nucleotide sequence of the specific molecular target for detecting green aerosol fungi in this example is shown in SEQ ID NO: 1.

[0038] 2. Example of primers for detecting specific molecular targets of green balloon bacteria

[0039] Example 2

[0040] The primers for detecting the specific molecular target of green balloon fungus in this embodiment include a forward primer AU-F and a reverse primer AU-R; the nucleotide sequence of the forward primer AU-F is shown in SEQ ID NO: 2, and the nucleotide sequence of the reverse primer AU-R is shown in SEQ ID NO: 3.

[0041] 3. Example of a kit for detecting green balloon bacteria

[0042] Example 3

[0043] The kit for detecting green balloon fungi of this embodiment includes the forward primer, reverse primer, ddH2O, and 2×Es Taq Master Mix in Example 2.

[0044] 4. Embodiment of the method for detecting green balloon bacteria

[0045] Example 4

[0046] The method for detecting green balloon fungus of the present embodiment comprises the following steps:

[0047] (1) Extract DNA from duck samples by boiling method;

[0048] (2) The DNA was amplified using the primers in Example 2 to obtain an amplified product and stored at 4°C. The reaction system (20 μL) for PCR amplification was: 7 μL of ddH2O, 10 μL of 2×Es Taq Master Mix, 0.5 μL of forward primer, 0.5 μL of reverse primer and 2 μL of template DNA, wherein the concentrations of the forward primer and the reverse primer were both 10 μmol / L; the reaction conditions for PCR amplification were: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 30 s, denaturation, annealing and extension were repeated for a total of 30 cycles; final extension at 72°C for 10 min;

[0049] (3) 7 μL of the amplified product was subjected to 2% agarose gel electrophoresis analysis. The electrophoresis analysis results showed that the size of the amplified fragment was about 312 bp, indicating that the sample to be tested contained duck-derived green aerosol bacteria.

[0050] Example 5

[0051] The method for detecting green balloon fungi in this embodiment is different from that in embodiment 4 only in that the samples to be tested in this embodiment are from pigs, chickens, sheep, mice, cattle, shrimps and crabs, respectively, and the contents not mentioned are carried out with reference to the steps in embodiment 4.

[0052] V. Experimental Examples

[0053] 1.1 Electrophoresis experiment

[0054] The genomic DNA of duck-derived green aerosol bacteria and control strains (Riemerella anatipestifer, Staphylococcus aureus, Escherichia coli, Salmonella, Streptococcus, Pasteurella) was extracted as templates, and amplified using the primers and PCR conditions in Example 4 to obtain amplified products. Then, 7 μL of the amplified products were analyzed by 2% agarose gel electrophoresis. The electrophoresis results are shown in FIG. Figure 1 As shown, M is DL2000 DNA Marker, 1-3 are duck Murray's bacillus, 4-6 are Staphylococcus aureus, 7-9 are Escherichia coli, 10-12 are Salmonella, 13-15 are Streptococcus, 16-17 are Pasteurella, and 18-21 are green balloon bacteria from sheep, green balloon bacteria from duck, green balloon bacteria from chicken, and green balloon bacteria from pig, respectively.

[0055] from Figure 1 It can be seen that the fragment size of the amplified product of duck-derived green aerosol bacteria is about 312 bp, while Riemerella anatipestifer, Staphylococcus aureus, Escherichia coli, Salmonella, Streptococcus, and Pasteurella did not amplify specific bands, indicating that the present invention can identify duck-derived green aerosol bacteria.

[0056] 1.2 Sensitivity experiment

[0057] The duck-derived green balloon fungus solution was diluted 10 times in a gradient, with a concentration range of 1×10 1 ~1×10 8 CFU / mL, DNA was extracted by boiling method, amplified using primers and PCR conditions in Example 4, and then the amplified product was analyzed by electrophoresis. The results are as follows Figure 2 As shown, M is DL2000 DNA Marker, 1:1×10 4 CFU / mL, 2: 1×10 5 CFU / mL, 3: 1×10 6 CFU / mL, 4: 1×10 7 CFU / mL, 5: 1×10 8 CFU / mL, 6: 1×10 1 CFU / mL, 7: 1×10 2 CFU / mL, 8: 1×10 3 CFU / mL.

[0058] from Figure 2 As can be seen, the bacterial concentration is 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL and 1×10 8CFU / mL electrophoresis results showed specific bands, and when the bacterial concentration was less than 1×10 4 CFU / mL, no specific bands appeared in the electrophoresis results. Therefore, the detection limit of duck-derived green balloon bacteria was 1×10 4 CFU / mL.

[0059] 1.3 Annealing temperature optimization test

[0060] The annealing temperature was optimized with reference to Example 4, and 11 temperature gradients were set, namely 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, and 60°C. The corresponding band amplification results are shown in Figure 3 shown.

[0061] Depend on Figure 3 It can be seen that the PCR detection effect is best at 57°C annealing temperature. <110> Henan College of Animal Husbandry and Economics <120> A specific molecular target, primer, kit and method for detecting green balloon bacteria <170> PatentIn version 3.5 <211> 312 <212> DNA <213> Green balloon fungus <221> Specific molecular targets <222> (1)..(312) <400> 1 taagccgtgg gctttcacat cagacttaag aaaccacctg cgctcccttt acgcccaata 60 aatccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta gttagccgtg 120 gctttctgat aagataccgt caagactgta gcagttatactc tacaatttgt tcttctctta 180 taacagagtt ttacgacccg aaggccttct tcactcacgc ggcattgctc cgtcaggctt 240 tcgcccattg cggaagattc cctactgctg cctcccgtag gagtttgggc cgtgtctcag 300 tcccaatgtgc 312 <211> 20 <212> DNA <213> Artificial sequence <221> Forward Primer <222> (1)..(20) <400> 2 taagccgtgg gctttcacat 20 <211> 20 <212> DNA <213> Artificial sequence <221> Reverse primer <222> (1)..(20) <400> 3 gccacattgg gactgagaca 20

Claims

1. A specific molecular target for detecting green balloon bacteria, characterized in that: The nucleotide sequence of the specific molecular target is shown in SEQ ID NO:

1.

2. A primer for detecting a specific molecular target of the green balloon fungus as claimed in claim 1, characterized in that: The nucleotide sequence of the forward primer is shown in SEQ ID NO:2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:

3.

3. A kit for detecting green balloon bacteria, characterized in that: Comprising the primer as claimed in claim 2.

4. The kit for detecting green balloon bacteria according to claim 3, characterized in that, The kit also includes ddH2O and 2×Es Taq Master Mix.

5. A method for detecting green balloon fungi, characterized in that: The following steps are involved: Extracting DNA from the sample to be tested, amplifying the DNA with the primers as claimed in claim 2 to obtain an amplified product, and performing electrophoresis analysis on the amplified product to determine whether there is a specific band of green balloon fungi; if the specific band exists, it indicates that the sample to be tested contains green balloon fungi, otherwise, it indicates that the sample to be tested does not contain green balloon fungi; The method is non-disease diagnosis and treatment.

6. The method for detecting green balloon bacteria according to claim 5, characterized in that, The amplification reaction system includes 7 μL of ddH2O, 10 μL of 2×Es Taq Master Mix, 0.5 μL of the forward primer, 0.5 μL of the reverse primer and 2 μL of template DNA; the concentrations of the forward primer and the reverse primer are both 10 μmol / L.

7. The method for detecting green balloon bacteria according to claim 6, characterized in that, The reaction conditions of the amplification are as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 50-60°C for 30 s, extension at 72°C for 30 s, denaturation, annealing and extension repeated for 30 cycles; final extension at 72°C for 10 min.

8. The method for detecting green balloon bacteria according to claim 7, characterized in that, The annealing temperature is 57°C.

9. The method for detecting green balloon bacteria according to any one of claims 5 to 8, characterized in that: The green balloon fungus is sourced from animal husbandry animals.

10. The method for detecting green balloon bacteria according to claim 9, characterized in that: The animal husbandry is one of duck, pig, chicken, sheep and cattle.

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