Multiplex PCR (Polymerase Chain Reaction) detection primer group, kit and detection method for pathogenic bacteria

Abstract

The invention discloses a multiplex PCR (Polymerase Chain Reaction) detection primer group, kit and detection method for pathogenic bacteria, and relates to the technical field of genetic molecules, and the method comprises the following steps: (1) designing primers; (2) extracting whole genome DNA of bacteria; (3) screening of primers of Rosella rosea, Acinetobacter loffithii and Aeromonas veronii, and verification of annealing temperature; and (4) carrying out multiple PCR primer screening on Rosella rosea, Acinetobacter luffae and Aeromonas veronii, and carrying out annealing temperature verification. According to the multiplex PCR detection primer group, the kit and the detection method for the pathogenic bacteria, a set of specific detection primer group is designed and synthesized by comparing and analyzing genomes of the three pathogenic bacteria, a multiplex PCR detection method for detecting genes of the three pathogenic bacteria is established, and the kit containing the specific primer group is assembled. The kit disclosed by the invention has the advantages of high specificity, no cross reaction with other bacteria, rapidness, simplicity, high sensitivity and the like when being used for detecting the three pathogenic bacterium genes.

Description

Technical Field

[0001] The present invention relates to the field of genetic molecular technology, and in particular to a primer set, a kit and a detection method for multiple PCR detection of pathogenic bacteria. Background Art

[0002] Acinetobacter lofyi, Roseburia muris, and Aeromonas veronii are all important pathogens of human, animal, and aquatic organisms. Acinetobacter lofyi is commonly found in soil, water, or dry environments. It is a potential pathogen for animals or humans with low immunity. It can not only cause hospital infections, sepsis, pneumonia, meningitis, urinary tract infections, skin wound infections, or acute gastroenteritis, but also reports of infections in rodents, poultry, and aquatic animals have increased in recent years, such as the death of mice and ducks infected with Acinetobacter lofyi, the isolation of Acinetobacter lofyi from raw milk of dairy cows, the death of rabbits infected with Acinetobacter lofyi, and the widespread occurrence of skin ulcers and internal bleeding in schizothorax in Sichuan Province due to infection with Acinetobacter lofyi from 2016 to 2017. Roseburia muris can be transmitted through the air and is commonly found in the upper respiratory tract of birds, geese, dogs, ducks, mice, and humans. It can seriously cause clinical symptoms of feather loss in waterfowl. With the increase in research reports on this bacterium, it was found that the bacterium often shows a high level of resistance to multiple commonly used antibiotics, and can be transmitted between humans and animals, resulting in reduced or ineffective efficacy of antibiotics against the bacterium, reduced infection cure rate, increased mortality rate, significantly prolonged the course of disease and treatment time, and greatly increased treatment costs. Therefore, it should be given enough attention. Aeromonas veronii can be transmitted through water sources, food sources, and wound infections, posing a serious threat to human health. It can cause diseases such as diarrhea, pneumonia, and sepsis in humans. At the same time, the bacterium is also the main pathogen of aquatic animals such as fish, shrimps, crabs, and turtles. It can cause ulcers on the surface of aquatic animals, congestion and bleeding of organs such as the liver and kidneys, and in severe cases, explosive death may occur. Aeromonas veronii has a strong adaptability to the environment. It is not only widely present in aquatic animals, but is also often detected in livestock and poultry meat, agricultural products, tap water, etc. In recent years, Aeromonas has become an important pathogenic bacterium for domestic infection. In view of the serious harm posed by Aeromonas veronii to food safety, some countries have listed it as a quarantine object for food safety and water quality. Therefore, the pathogenicity of this bacterium should also attract sufficient attention.

[0003] At present, the methods for detecting Roseburia murinus, Acinetobacter lofyi, and Aeromonas veronii include 16s rRNA sequencing, whole genome sequencing, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identification. 16s rRNA sequencing and whole genome sequencing have long detection time and high cost; matrix-assisted laser desorption ionization time-of-flight mass spectrometry identification uses a relatively expensive mass spectrometer and is troublesome to maintain, so it is not conducive to popularization and use in grassroots units. In order to solve the above problems, the present invention designs and synthesizes a set of specific detection primer sets, and establishes a multiplex PCR detection method for detecting three pathogenic bacteria genes. Summary of the invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to solve the problems raised by the background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions: a multiplex PCR detection primer set, a kit and a detection method for pathogenic bacteria, which include the following methods:

[0006] ①: Primer design;

[0007] ②: Extraction of bacterial whole genome DNA;

[0008] ③: Primer screening and annealing temperature verification for Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii primers;

[0009] ④: Screening of multiple PCR primers and verification of annealing temperature for Roseburia murinus, Acinetobacter lofi, and Aeromonas veronii;

[0010] ⑤: Multiple PCR cross-validation of Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii.

[0011] ⑥: Establishment of multiplex PCR detection kit for Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii.

[0012] ⑦: Evaluation of the specificity and sensitivity of the multiplex PCR gene detection kit for Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii.

[0013] Preferably, the design steps based on ① are as follows:

[0014] S1: Download all strain genome files and genome annotation files of the target species from the NCBI genbank database;

[0015] S2: NCBI genbank database genome files are divided into reference genomes and common strain genomes;

[0016] S3: break the reference genome sequence into 1000 bp in length;

[0017] S4: Common strain genome construction comparison database, align the 1000bp kmer sequence obtained in the previous step to the genomes of all common strains;

[0018] S5: Align the potential conserved region sequences on the NT database using ncbi-blast;

[0019] S6: Using the conserved sequence as a template, primers were designed using primer3;

[0020] S7: Verify the specificity of the amplified sequence again;

[0021] S8: Select the optimal primers for each bacterial species based on the results.

[0022] Preferably, in the method ②, each of the Roseburia murinsis, Acinetobacter lofyi and Aeromonas veronii strains is inoculated into a TSB liquid culture medium containing fetal bovine serum.

[0023] Preferably, the method based on ③ adopts primer screening for gene amplification of Roseburia muris and verification of annealing temperature, primer screening for gene amplification of Acinetobacter loffei and verification of annealing temperature, and primer screening for gene amplification of Aeromonas veronii and verification of annealing temperature.

[0024] Preferably, the method based on ④ uses multiple PCR primer combination verification of Roseburia muris, Acinetobacter loffei, Aeromonas veronii, multiple PCR primer combination 1 PCR amplification verification, multiple PCR primer combination 2 PCR amplification verification, multiple PCR primer combination 3 PCR amplification verification and multiple PCR primer combination 4 PCR amplification verification.

[0025] Preferably, based on the screening of multiple PCR primers for Roseburia muris, Acinetobacter lofyi, and Aeromonas veronii in ⑤, we finally obtain a set of multiple PCR primer sets for Roseburia muris, Acinetobacter lofyi, and Aeromonas veronii with good sensitivity and specificity.

[0026] Preferably, the PCR reaction system based on ⑥ is 20 μL: 3 μL of pathogenic bacteria DNA genome; 5 μL of equal volumes of upstream and downstream primers of multiplex PCR combination 1, and 6 μL is aspirated; 10 μL of Taq DNA polymerase; 6 μL of sterile ddH2O. Amplification reaction procedure: 95°C pre-denaturation for 4 min; 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 30 s, a total of 32 cycles; 72°C final extension for 7 min.

[0027] Preferably, the method according to ⑦ comprises the sensitivity test of the multiple PCR gene detection kit for Acinetobacter lofyi, Aeromonas veronii, and Roseburia murinsis, the multiple PCR specificity verification 1 of Acinetobacter lofyi, Aeromonas veronii, and Roseburia murinsis, and the multiple PCR specificity verification 2 of Acinetobacter lofyi, Aeromonas veronii, and Roseburia murinsis.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The pathogenic bacteria multiple PCR detection primer set, kit and detection method are designed and synthesized by comparing and analyzing the genomes of three pathogenic bacteria, and a multiple PCR detection method for detecting the genes of three pathogenic bacteria is established, and a kit containing the specific primer set is assembled. The detection of the three pathogenic bacteria genes using the kit of the present invention has the advantages of strong specificity, no cross reaction with other bacteria, rapidity and simplicity, good sensitivity, etc., and can detect a large number of samples at the same time, with low cost, and thus has the advantages of simplicity, high sensitivity, short detection time and good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention is further described below in conjunction with the accompanying drawings and embodiments:

[0031] Table 1 is a schematic diagram of the structures of PCR amplification primers for Acinetobacter lofi, Aeromonas veronii, and Roseburia murinus of the present invention;

[0032] Table 2 is a schematic diagram of the structure of the target sequence information amplified by PCR primers of Acinetobacter lofi, Aeromonas veronii, and Roseburia murinus of the present invention;

[0033] Table 3 is a schematic diagram of the structure of the multiplex PCR primer combination of the present invention;

[0034] Table 4 is a schematic diagram of the structure of the mixed grouping of three pathogens of the present invention;

[0035] Figure 1 It is a schematic diagram of the amplification results of the primer 1 of R. muris of the present invention at different annealing temperatures (M: DL2000, 1-7: from left to right, the amplification results of the primer annealing temperatures of 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and 60°C for R. muris);

[0036] Figure 2 It is a schematic diagram of the amplification results of the primer 2 of R. muris of the present invention at different annealing temperatures (M: DL2000, 1-8: from left to right, the amplification results of the primer annealing temperatures of 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and 60°C for R. muris);

[0037] Figure 3It is a schematic diagram of the amplification results of the primer 3 of R. muris of the present invention at different annealing temperatures (M: DL2000, 1-8: from left to right, the amplification results of the primer annealing temperatures of 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and 60°C for R. muris);

[0038] Figure 4 It is a schematic diagram of the amplification results of the Acinetobacter lofyi primer 1 of the present invention at different annealing temperatures (M: DL2000, 1-8: from left to right, the amplification results of the Acinetobacter lofyi primer annealing temperatures of 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and 60°C);

[0039] Figure 5 It is a schematic diagram of the amplification results of the Acinetobacter lofyi primer 2 of the present invention at different annealing temperatures (M: DL2000, 1-8: from left to right, the amplification results of the Acinetobacter lofyi primer annealing temperatures of 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and 60°C);

[0040] Figure 6 It is a schematic diagram of the amplification results of the Acinetobacter lofyi primer 3 of the present invention at different annealing temperatures (M: DL2000, 1-8: from left to right, the amplification results of the Acinetobacter lofyi primer annealing temperatures of 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and 60°C);

[0041] Figure 7 It is a schematic diagram of the structure of the amplification results of Aeromonas veronii primer 1 at different annealing temperatures of the present invention (M: DL2000, 1-8: from left to right, the amplification results of Aeromonas veronii primer annealing temperatures of 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and 60°C respectively);

[0042] Figure 8 It is a schematic diagram of the amplification results of Aeromonas veronii primers 2, 3, and 4 at different annealing temperatures of the present invention (M: DL2000, 1-8: from left to right, the amplification results of Aeromonas veronii primers at annealing temperatures of 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and 60°C, respectively);

[0043] Fig. 9 It is a schematic diagram of the amplification results of the primer set 1 of three pathogens of the present invention at different annealing temperatures (M: DL2000, 1-8: from left to right, the amplification results of the multiplex PCR of three pathogens at annealing temperatures of 52°C, 54°C, 56°C, 58°C, and 60°C, respectively);

[0044] Fig.10It is a schematic diagram of the amplification results of the primer set 2 for three pathogens of the present invention at different annealing temperatures (M: DL2000, 1-8: from left to right, the amplification results of the multiplex PCR of three pathogens at annealing temperatures of 54°C, 56°C, 58°C, 60°C, and 62°C, respectively);

[0045] Fig.11 It is a schematic diagram of the amplification results of primer set 3 of three pathogens of the present invention at different annealing temperatures (M: DL2000, 1-8: from left to right, the amplification results of multiplex PCR of three pathogens at annealing temperatures of 50°C, 52°C, 54°C, 56°C, and 58°C respectively);

[0046] Fig.12 It is a schematic diagram of the amplification results of the primer set 4 of three pathogens of the present invention at different annealing temperatures (M: DL2000, 1-8: from left to right, the amplification results of the multiplex PCR of three pathogens at annealing temperatures of 54°C, 56°C, 58°C, 60°C, and 52°C, respectively);

[0047] Fig.13 Schematic diagram of the amplification results of the three pathogen primers of the present invention at different annealing temperatures (M: DL2000, 1: amplification result of mixed bacteria of group 1; 3: amplification result of mixed bacteria of group 2; 5: amplification result of mixed bacteria of group 31; 7: amplification result of mixed bacteria of group 4; 2, 4, 6, 8: negative controls);

[0048] Fig.14 It is a structural schematic diagram of the amplification results of the murine rhinitis Roseburia solution of different dilution gradients of the present invention (M: MarkerDL2000; 1-9: from left to right, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8, 10-9 dilution gradient bacterial solution amplification results);

[0049] Fig.15 It is a structural schematic diagram of the amplification results of Acinetobacter loffei liquid with different dilution gradients of the present invention (M: MarkerDL2000; 1-9: from left to right, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8, 10-9 dilution gradient bacterial liquid amplification results);

[0050] Fig.16 It is a structural schematic diagram of the amplification results of Aeromonas veronii liquid at different dilution gradients of the present invention (M: MarkerDL2000; 1-9: from left to right are 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8, 10-9 dilution gradient bacterial liquid amplification results);

[0051] Fig.17Schematic diagram of the structure of the specific verification of the multiplex kit of the present invention (M: DL Marker2000; 1: amplification result of Acinetobacter loffei; 2: amplification result of Roseburia murinus; 3: amplification result of Aeromonas veronii; 4: amplification result of Escherichia coli; 5: amplification result of Staphylococcus aureus; 6: amplification result of Salmonella; 7: amplification result of Streptococcus pneumoniae; 8: amplification result of Enterococcus faecalis; 9: amplification result of Haemophilus influenzae; 10: amplification result of Pasteurella multocida);

[0052] Fig.18 It is a structural schematic diagram of the amplification results of Acinetobacter Lofyi from different sources and different time periods of the present invention (M: DLMarker2000; 1: amplification results of Acinetobacter Lofyi isolated from duck tissue; 2: amplification results of Acinetobacter Lofyi isolated from duck tissue; 3: amplification results of Acinetobacter Lofyi isolated from cow tissue; 4: amplification results of Acinetobacter Lofyi isolated from fish tissue; 5: amplification results of Acinetobacter Lofyi isolated from mouse tissue; 6: amplification results of Acinetobacter Lofyi isolated from mouse tissue; 7: amplification results of Acinetobacter Lofyi isolated from chicken tissue);

[0053] Fig.19 It is a structural schematic diagram of the amplification results of Aeromonas Veronii from different sources and different time periods of the present invention (M: DLMarker2000; 1: amplification results of Aeromonas Veronii isolated from fish tissue; 2: amplification results of Aeromonas Veronii isolated from fish tissue; 3: amplification results of Aeromonas Veronii isolated from fish tissue; 4: amplification results of Aeromonas Veronii isolated from fish tissue; 5: amplification results of Aeromonas Veronii isolated from pig tissue; 6: amplification results of Aeromonas Veronii isolated from duck tissue);

[0054] Fig. 20 Schematic diagram of the amplification results of R. muris from different sources and different time periods of the present invention (M: DLMarker2000; 1: amplification results of R. muris isolated from goose tissue; 2: amplification results of R. muris isolated from mouse tissue; 3: amplification results of R. muris isolated from dog tissue; 4: amplification results of R. muris isolated from duck tissue). DETAILED DESCRIPTION

[0055] This section will describe in detail the specific embodiments of the present invention. The preferred embodiments of the present invention are shown in the accompanying drawings. The purpose of the accompanying drawings is to supplement the description of the text part of the specification with graphics, so that people can intuitively and vividly understand each technical feature and the overall technical solution of the present invention, but it cannot be understood as a limitation on the scope of protection of the present invention.

[0056] In the description of the present invention, it should be understood that descriptions involving orientations, such as up, down, front, back, left, right, etc., and orientations or positional relationships indicated are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0057] In the description of the present invention, "greater than", "less than", "exceed" etc. are understood as not including the number itself, and "above", "below", "within" etc. are understood as including the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0058] In the description of the present invention, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention based on the specific content of the technical solution.

[0059] See also Figure 1-4 The present invention provides a technical solution: a multiplex PCR detection primer set, a kit and a detection method for pathogenic bacteria, the operation is as follows:

[0060] 1. Primer design

[0061] Here are the steps:

[0062] ① Download all strain genome files and genome annotation files of the target species from the NCBI genbank database.

[0063] ②NCBI genbank database genome files are divided into reference genomes and common strain genomes.

[0064] ③ Break the reference genome sequence into segments of 1000 bp in length.

[0065] ④ Construct a comparison database for the genomes of common strains, and compare the 1000bp kmer sequence obtained in the previous step to the genomes of all common strains. This step screens kmer sequences that can be compared to more than 95% of the strains, indicating that the kmer sequence is a potential conserved region.

[0066] ⑤ Further compare the potential conservative region sequences on the NT database using ncbi-blast, and further screen the candidate template sequences with a consistency rate of less than 80% with non-target species. The candidate template sequences screened in this step are the targeted template sequences that meet the requirements of PCR identification for both conservation and specificity.

[0067] ⑥Using the conserved sequence as a template, primers were designed using primer3, with a primer length of 18-27 bp, a TM value of 50-62 degrees, a GC content of 40-60%, and an amplification length of 200-900 bp.

[0068] ⑦ Since the primer amplification region is a partial sequence of the conserved sequence, the specificity of the amplified sequence needs to be verified again. After the primer design is completed, the amplified sequence is further compared with the NT database using ncbi-blast to verify whether the amplified product sequence meets the consistency rate with non-target species and is less than 80%.

[0069] ⑧ According to the results, 3 pairs of optimal primers were screened for Acinetobacter lofi, 3 pairs of optimal primers were screened for Roseburia murinus, and 4 pairs of optimal primers were screened for Aeromonas veronii. The primer sequence information is shown in Table 1 below:

[0070]

[0071] The amplified sequence information of Table 1 is shown in Table 2 below:

[0072]

[0073]

[0074]

[0075] Table 2

[0076] 2. Extraction of bacterial whole genome DNA

[0077] The strains of Roseburia murinsis, Acinetobacter lofyi, and Aeromonas veronii were inoculated into TSB liquid culture medium containing fetal bovine serum respectively, and cultured at 37°C for 12 h. 1 mL of overnight cultured Roseburia murinsis, Acinetobacter lofyi, and Aeromonas veronii were collected after centrifugation at 12000 r / min for 2 min, 100 μL of sterile distilled water was added for shaking and mixing, and then boiled in a water bath for 10 min and ice bathed for 10 min; after centrifugation at 12000 r / min for 5 min, the supernatant was taken as the extracted whole genome DNA and stored for later use.

[0078] 3. Primer screening and annealing temperature verification for Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii primers.

[0079] 3.1 Primer screening and annealing temperature verification for Roseburia murinus gene amplification

[0080] PCR reaction system 20μL: 2μL of R. murinii genome template; 1uL of each of 3 pairs of upstream and downstream primers (5umoL); 10μL of TaqDNA polymerase; 6μL of sterile ddH2O. Amplification reaction procedure: 95℃ pre-denaturation for 4min; 95℃ denaturation for 30s, XX℃ annealing for 30s, 72℃ extension for 30s, a total of 32 cycles; 72℃ final extension for 7min. The annealing temperature was set to 48℃~60℃. The PCR amplification product was detected by 1% agarose gel electrophoresis and gel recovery, and the PCR gel recovery product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The results showed that primer 1 of R. murinii could amplify the target band at 418bp within the set annealing temperature range, without non-specific amplification, and the amplification effect was best when the annealing temperature was 54℃ and 56℃. Figure 1 As shown in the figure, the primer 2 of R. murinus did not amplify the target band within the set annealing temperature range. Figure 2 As shown in the figure, primer 3 of R. murinus did not amplify the target band within the set annealing temperature range, and there was non-specific amplification, such as Figure 3 shown.

[0081] 3.2 Primer screening and annealing temperature verification for Acinetobacter lofi

[0082] PCR reaction system 20μL: 2μL of Acinetobacter lofyi genome template; 1uL of each of 3 pairs of upstream and downstream primers (5umoL) of Acinetobacter lofyi; 10μL of Taq DNA polymerase; 6μL of sterile ddH2O. Amplification reaction procedure: 95℃ pre-denaturation for 4min; 95℃ denaturation for 30s, XX℃ annealing for 30s, 72℃ extension for 30s, a total of 32 cycles; 72℃ final extension for 7min. The annealing temperature was set to 48℃~60℃. The PCR amplification product was detected by 1% agarose gel electrophoresis and gel recovery, and the PCR gel recovery product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The results showed that Acinetobacter lofyi primer 1 could amplify the target band at 528bp within the set annealing temperature range of 50℃~60℃, without non-specific amplification, and the amplification effect was best when the annealing temperature was 56℃. Figure 4 As shown in the figure, the primer 2 of Acinetobacter lofyi can amplify the target band at 211bp within the set annealing temperature range of 50℃~60℃, without non-specific amplification. Figure 5 As shown in the figure, the primer 3 of Acinetobacter lofyi can amplify the target band at 372bp within the set annealing temperature range of 50℃~60℃, and non-specific bands are also amplified, such as Figure 6 shown.

[0083] 3.3 Primer screening and annealing temperature verification for Aeromonas veronii gene amplification

[0084] PCR reaction system 20μL: 2μL of Aeromonas veronii genome template; 1uL of each of 6 pairs of upstream and downstream primers of Aeromonas veronii (5umoL); 10μL of Taq DNA polymerase; 6μL of sterile ddH2O. Amplification reaction procedure: 95℃ pre-denaturation for 4min; 95℃ denaturation for 30s, XX℃ annealing for 30s, 72℃ extension for 30s, a total of 32 cycles; 72℃ final extension for 7min. The annealing temperature was set to 48℃~60℃. The PCR amplification product was detected by 1% agarose gel electrophoresis and gel recovery, and the PCR gel recovery product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The results showed that Aeromonas veronii primer 1 could amplify the target band at 329bp within the set annealing temperature range of 48℃~60℃, without non-specific amplification, and the best amplification effect at 329bp was achieved when the annealing temperature was 52℃, 54 and 56℃. Figure 7 As shown in the figure, the primer 2 of Aeromonas veronii can amplify the target band at 808bp at the set annealing temperature of 48℃~60℃, and the amplification effect is best when the annealing temperature is 54℃, 56℃, 58℃, and 60℃, and no specific band is amplified. Figure 8 As shown in the figure, the primer 3 of Aeromonas veronii has a large amount of non-specific amplification in the set annealing temperature range of 48°C to 60°C; the primer 4 of Aeromonas veronii can amplify the target band at 360bp in the set annealing temperature range of 48°C to 60°C, and both have non-specific amplification, such as Figure 8 shown.

[0085] 4. Screening of multiple PCR primers and verification of annealing temperature for Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii.

[0086] 4.1 Multiple PCR primer combinations for R. muris, Acinetobacter loffei, and Aeromonas veronii Based on the screening and annealing temperature verification results of single PCR primers for R. muris, Acinetobacter loffei, and Aeromonas veronii, we can screen out primers with good specificity and sensitivity: R. muris primers 1F / R; Acinetobacter loffei primers 1F / R and 2F / R; Aeromonas veronii primers 1F / R and F / R2. The selected primer combinations were combined and PCR verified, as shown in Table 3 below:

[0087]

[0088]

[0089] Table 3

[0090] 4.2 Multiplex PCR primer combination 1 PCR amplification verification

[0091] After mixing equal volumes of liquid bacterial liquid of R. muris, Acinetobacter lofyi, and Aeromonas veronii cultured overnight, extract genomic DNA from the mixed bacterial liquid according to the steps of 2.1 bacterial whole genome DNA extraction. PCR reaction system 20μL: 3μL of mixed genome of R. muris, Acinetobacter lofyi, and Aeromonas veronii; 6uL of mixed liquid of equal volumes of upstream and downstream primers (5umoL) of multiple PCR combination 1; 10μL of Taq DNA polymerase; 4μL of sterile ddH2O. Amplification reaction procedure: 95℃ pre-denaturation for 4min; 95℃ denaturation for 30s, XX℃ annealing for 30s, 72℃ extension for 30s, a total of 32 cycles; 72℃ final extension for 7min. According to the annealing temperature results of the corresponding primers of the three pathogens explored, the multiple PCR annealing temperature range was set to 52℃~60℃. PCR amplification products were detected by 1% agarose gel electrophoresis. The results showed that the primer set of multiplex PCR combination 1 could amplify the target band within the set annealing temperature range without non-specific amplification, and the amplification effect was best when the annealing temperature was 56°C. Fig. 9 shown.

[0092] 4.2 Multiplex PCR primer combination 2PCR amplification verification

[0093] After mixing equal volumes of the liquid bacterial liquid of R. muris, Acinetobacter lofyi, and Aeromonas veronii cultured overnight, extract the genomic DNA of the mixed bacterial liquid according to the steps of 2.1 bacterial whole genome DNA extraction. PCR reaction system 20μL: 3μL of mixed genome of R. muris, Acinetobacter lofyi, and Aeromonas veronii; 6uL of mixed liquid of 2 upstream and downstream primers (5umoL) of multiple PCR combination; 10μL of Taq DNA polymerase; 4μL of sterile ddH2O. Amplification reaction program: 95℃ pre-denaturation for 4min; 95℃ denaturation for 30s, XX℃ annealing for 30s, 72℃ extension for 30s, a total of 32 cycles; 72℃ final extension for 7min. According to the annealing temperature exploration results of the corresponding primers of the three pathogens, the multiple PCR annealing temperature range was set to 54℃~62℃. PCR amplification products were detected by 1% agarose gel electrophoresis. The results showed that in addition to the target band, multiple PCR combination 2 primer sets had multiple nonspecific amplifications, such as Fig.10 shown.

[0094] 4.3 Multiplex PCR primer combination 3PCR amplification verification

[0095] After mixing equal volumes of the liquid bacterial liquid of R. muris, Acinetobacter lofyi, and Aeromonas veronii cultured overnight, extract the genomic DNA of the mixed bacterial liquid according to the steps of 2.1 bacterial whole genome DNA extraction. PCR reaction system 20μL: 3μL of mixed genome of R. muris, Acinetobacter lofyi, and Aeromonas veronii; 3 equal volumes of upstream and downstream primers (5umoL) of multiple PCR combination, 6uL of mixed solution; 10μL of Taq DNA polymerase; 4μL of sterile ddH2O. Amplification reaction program: 95℃ pre-denaturation for 4min; 95℃ denaturation for 30s, XX℃ annealing for 30s, 72℃ extension for 30s, a total of 32 cycles; 72℃ final extension for 7min. According to the annealing temperature exploration results of the corresponding primers of the three pathogens, the multiple PCR annealing temperature range was set to 50℃~58℃. The PCR amplification products were detected by 1% agarose gel electrophoresis. The results showed that the primers of the multiplex PCR combination 3 could amplify the target bands of Roseburia murinus and Aeromonas veronii within the set annealing temperature range, while the primers of Acinetobacter lofyi could not amplify the target bands within the set annealing temperature range. Fig.11 shown.

[0096] 4.4 Multiplex PCR primer combination 4PCR amplification verification

[0097] After mixing equal volumes of the liquid bacterial liquid of R. muris, Acinetobacter lofyi, and Aeromonas veronii cultured overnight, extract the genomic DNA of the mixed bacterial liquid according to the steps of 2.1 bacterial whole genome DNA extraction. PCR reaction system 20μL: 3μL of mixed genome of R. muris, Acinetobacter lofyi, and Aeromonas veronii; 6uL of mixed liquid of 4 upstream and downstream primers (5umoL) of multiple PCR combination; 10μL of Taq DNA polymerase; 4μL of sterile ddH2O. Amplification reaction program: 95℃ pre-denaturation for 4min; 95℃ denaturation for 30s, XX℃ annealing for 30s, 72℃ extension for 30s, a total of 32 cycles; 72℃ final extension for 7min. According to the annealing temperature exploration results of the corresponding primers of the three pathogens, the multiple PCR annealing temperature range was set to 54℃~62℃, and the PCR amplification products were detected by 1% agarose gel electrophoresis. The results showed that the multiplex PCR combination of 4 primers could amplify the target bands of Roseburia murinus, Aeromonas veronii, and Acinetobacter lofi within the set annealing temperature range, and there was non-specific amplification, such as Fig.12 shown.

[0098] 5. Multiple PCR cross-validation of Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii.

[0099] Based on the screening of multiple PCR primers for Roseburia muris, Acinetobacter lofyi, and Aeromonas veronii, we finally obtained a set of multiple PCR primers set 1 for Roseburia muris, Acinetobacter lofyi, and Aeromonas veronii with good sensitivity and specificity.

[0100] Grouping Pathogen mixture 1 Roseburia murinus, Acinetobacter lofi, Aeromonas veronii 2 Roseburia murinus, Acinetobacter lofi 3 Aeromonas veronii, Roseburia murinus 4 Acinetobacter lofivi, Aeromonas veronii

[0101] Table 4

[0102] The liquid bacterial liquid of Roseburia murinsis, Acinetobacter lofyi, and Aeromonas veronii cultured overnight was mixed in equal volumes according to the grouping shown in Table 4 above, and the genomic DNA of the mixed bacterial liquid was extracted according to the bacterial whole genome DNA extraction process in step 2. PCR reaction system 20μL: 3μL of mixed genome of Roseburia murinsis, Acinetobacter lofyi, and Aeromonas veronii; equal volumes of upstream and downstream primers (5umoL) of multiple PCR combination 1, and 6uL was aspirated; 10μL of Taq DNA polymerase; 4μL of sterile ddH2O. Amplification reaction procedure: pre-denaturation at 95℃ for 4min; denaturation at 95℃ for 30s, annealing at 56℃ for 30s, extension at 72℃ for 30s, for a total of 32 cycles; final extension at 72℃ for 7min. The amplified products were detected by 1% agarose gel electrophoresis. The results showed that the corresponding target bands could be amplified for each group of mixed pathogens, without non-specific amplification, and there were no non-specific bands in the negative controls, such as Fig.13 shown.

[0103] 6. Establishment of a multiplex PCR detection kit for Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii.

[0104] PCR reaction system 20μL: 3μL pathogenic bacteria DNA genome; Multiplex PCR combination 1 upstream and downstream primers (5umoL) are mixed in equal volumes, and 6uL is drawn; Taq DNA polymerase 10μL; Sterile ddH2O 6μL. Amplification reaction program: 95℃ pre-denaturation for 4min; 95℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 30s, a total of 32 cycles; 72℃ final extension for 7min. Take 7ul of the amplified product for 1% agarose gel electrophoresis detection. If the amplified band is around 329bp, it is judged to be positive for Aeromonas veronii; if the amplified band is around 528bp, it is judged to be positive for Acinetobacter lofyi; if the amplified band is around 418bp, it is judged to be positive for Roseburia murinus; if there is no amplified band, it is judged to be negative.

[0105] 7. Evaluation of the specificity and sensitivity of the multiplex PCR gene detection kit for Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii.

[0106] 7.1 Sensitivity test of multiple PCR gene detection kit for Acinetobacter lofi, Aeromonas veronii and Roseburia murinus

[0107] The positive bacteria of Roseburia muris, Acinetobacter loffei, and Aeromonas veronii were inoculated into TSB liquid culture medium containing bovine serum, and the bacteria were enriched at 37°C overnight. 1 mL of the bacterial solution was taken for each, and the bacterial solution was diluted 10 times in a gradient manner, and the dilution degrees were 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8, and 10-9. The genomic DNA of the bacterial solution was extracted according to the whole genome DNA extraction process of bacteria in step 2, and PCR was performed according to the instructions for use of the kit. The sensitivity of the kit was determined based on the test results. The results showed that the minimum detection amount of the Roseburia muris gene in the multiple PCR system established by the present invention was 7×103CFU / mL; the minimum detection amount for Acinetobacter loffei was 7×104CFU / mL, and the minimum detection amount for Aeromonas veronii was 8×104CFU / mL (such as Fig.14 , 15 , as shown in 16).

[0108] Multiplex PCR specificity validation of Acinetobacter lofi, Aeromonas veronii, and Roseburia murinus 1

[0109] The genomic DNA of Acinetobacter loffei, Aeromonas veronii, Roseburia murinus, Escherichia coli, Staphylococcus, Salmonella, Streptococcus pneumoniae, Enterococcus faecalis, Haemophilus influenzae, and Pasteurella multocida isolated from mice, ducks, cows, and rabbits stored in the laboratory was used for PCR amplification. The PCR was performed according to the instructions of the kit. The results showed that the amplified product of Acinetobacter loffei had a target band at around 528 bp, the product of Aeromonas veronii had a target band at around 418 bp, and the product of Roseburia murinus had a target band at around 329 bp. No amplified bands were found in other strains, such as Fig.17 shown.

[0110] Multiplex PCR specificity validation of Acinetobacter lofi, Aeromonas veronii, and Roseburia murinus 2

[0111] Seven strains of Acinetobacter lofyi isolated from mouse, duck, chicken, cow, and fish tissues at different time periods; six strains of Aeromonas veronii isolated from fish, pig, and duck tissues at different time periods; and four strains of Roseburia murinus isolated from goose, dog, duck, and mouse tissues were collected and cultured, and genomic DNA was extracted for PCR amplification. PCR was performed according to the instructions of the kit. The results showed that the target band appeared at around 528 bp in the amplified product of Acinetobacter lofyi, such as Fig.18 As shown; the target band of Aeromonas veronii product appears at around 418 bp, such as Fig.19 As shown in the figure, the target band appeared at about 329 bp in the product of Roebuckella murinus, and no amplified bands were found in other strains, such as Fig. 20 shown.

[0112] The embodiments of the present invention are described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the technical field without departing from the purpose of the present invention.

Claims

1. A primer set, a kit and a detection method for multiplex PCR detection of pathogenic bacteria, characterized in that: The method includes the following: ①: Primer design; ②: Extraction of bacterial whole genome DNA; ③: Primer screening and annealing temperature verification for Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii primers; ④: Screening of multiple PCR primers and verification of annealing temperature for Roseburia murinus, Acinetobacter lofi, and Aeromonas veronii; ⑤: Multiple PCR cross-validation of Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii. ⑥: Establishment of multiplex PCR detection kit for Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii. ⑦: Evaluation of the specificity and sensitivity of the multiplex PCR gene detection kit for Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii.

2. The multiplex PCR detection primer set, kit and detection method for pathogenic bacteria according to claim 1, characterized in that: The design steps based on ① are as follows: S1: Download all strain genome files and genome annotation files of the target species from the NCBI genbank database; S2: NCBI genbank database genome files are divided into reference genomes and common strain genomes; S3: break the reference genome sequence into 1000 bp in length; S4: Common strain genome construction comparison database, align the 1000bp kmer sequence obtained in the previous step to the genomes of all common strains; S5: Align the potential conserved region sequences on the NT database using ncbi-blast; S6: Using the conserved sequence as a template, primers were designed using primer3; S7: Verify the specificity of the amplified sequence again; S8: Select the optimal primers for each bacterial species based on the results.

3. The multiplex PCR detection primer set, kit and detection method for pathogenic bacteria according to claim 2, characterized in that: Based on ②, the strains of Roseburia murinus, Acinetobacter lofivi, and Aeromonas veronii were inoculated into TSB liquid culture medium containing fetal bovine serum.

4. The multiplex PCR detection primer set, kit and detection method for pathogenic bacteria according to claim 3, characterized in that: The method based on ③ adopts the screening of gene amplification primers for Roseburia muris and the verification of annealing temperature, the screening of gene amplification primers for Acinetobacter loffei and the screening of gene amplification primers for Aeromonas veronii and the verification of annealing temperature.

5. The multiplex PCR detection primer set, kit and detection method for pathogenic bacteria according to claim 4, characterized in that: The method based on ④ uses multiple PCR primer combination verification of Roseburia murinus, Acinetobacter loffei, and Aeromonas veronii, multiple PCR primer combination 1 PCR amplification verification, multiple PCR primer combination 2 PCR amplification verification, multiple PCR primer combination 3 PCR amplification verification, and multiple PCR primer combination 4 PCR amplification verification.

6. The multiplex PCR detection primer set, kit and detection method for pathogenic bacteria according to claim 5, characterized in that: Based on the screening of multiple PCR primers for Roseburia muris, Acinetobacter lofyi, and Aeromonas veronii in ⑤, we finally obtained a set of multiple PCR primer sets for Roseburia muris, Acinetobacter lofyi, and Aeromonas veronii with good sensitivity and specificity.

7. The multiplex PCR detection primer set, kit and detection method for pathogenic bacteria according to claim 6, characterized in that: The PCR reaction system based on ⑥ is 20 μL: 3 μL of pathogenic bacteria DNA genome; 5 μL of equal volumes of upstream and downstream primers of multiple PCR combination 1, and 6 μL is drawn; 10 μL of Taq DNA polymerase; 6 μL of sterile ddH2O. Amplification reaction procedure: 95℃ pre-denaturation for 4 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, a total of 32 cycles; 72℃ final extension for 7 min.

8. The multiplex PCR detection primer set, kit and detection method for pathogenic bacteria according to claim 7, characterized in that: The method is based on the sensitivity test of the multiple PCR gene detection kit for Acinetobacter lofyi, Aeromonas veronii, and Roseburia murinsis in ⑦, the multiple PCR specificity verification 1 of Acinetobacter lofyi, Aeromonas veronii, and Roseburia murinsis, and the multiple PCR specificity verification of Acinetobacter lofyi, Aeromonas veronii, and Roseburia murinsis.

Images