Rapid detection kit and detection method for detecting flavobacterium psychrophilum

Abstract

The invention discloses a rapid detection kit and a detection method for detecting flavobacterium psychrophilum, and relates to the technical field of molecular biology. The method comprises the following steps: firstly, obtaining whole genome sequences of flavobacterium psychrophilum through a high-throughput sequencing technology, comparing the whole genome sequences one by one in an NCBI (National Center of Biotechnology Information) database to find out specific sequences which are completely not matched with other genomes, designing specific primers according to the specific sequences, and detecting the specificity of the primers through other strains, so as to identify the flavobacterium psychrophilum. And finally, the primer with high specificity is screened out. The method comprises the following steps of: firstly, designing a multiple PCR primer pair combination of the flavobacterium psychrophilum, and performing multiple PCR detection by utilizing the multiple PCR primer pair combination, so that a result shows that the multiple PCR primer pair combination designed and screened by the method has a good amplification effect on the detected target flavobacterium psychrophilum, and has remarkable specificity and relatively high detection sensitivity when being used for detecting the flavobacterium psychrophilum.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly to a rapid detection kit and a detection method for detecting Flavobacterium psychrophilum. Background Art

[0002] Flavobacterium psychrophilum belongs to the genus Flavobacterium of the phylum Bacteroidetes, and is a psychrophilic pathogen that often grows and multiplies at 4-23°C, with the optimal growth temperature being 15-18°C.

[0003] With the development of molecular biology techniques, the PCR technique has been widely applied in the field of rapid detection of pathogens. The multiplex PCR technique is based on the conventional PCR technique. By mixing multiple pairs of primers and optimizing the reaction conditions, the purpose of jointly detecting multiple pairs of primers for one bacterium is achieved, greatly improving the detection efficiency and the accuracy of the detection results. At the same time, it retains the characteristics of high sensitivity and specificity of ordinary PCR. The present invention intends to develop multiple specific primers for Flavobacterium psychrophilum and a multiplex PCR detection method to improve the specificity, sensitivity, and accuracy of the detection of Flavobacterium psychrophilum. Summary of the Invention

[0004] The purpose of the present invention is to provide a rapid detection kit and a detection method for detecting Flavobacterium psychrophilum to solve the problems existing in the above-mentioned prior art and improve the specificity, sensitivity, and accuracy of the detection of Flavobacterium psychrophilum.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides a multiplex PCR primer pair combination for detecting Flavobacterium psychrophilum, and the multiplex PCR primer pair combination is composed of primer pairs 2FP90-F / R, FP1024-F / R, and 2FP1022-F / R;

[0007] The FP1024-F / R includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.6;

[0008] The 2FP1022-F / R includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.11 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.12.

[0009] The 2FP90-F / R includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.9 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.10.

[0010] The present invention also provides the application of the above-mentioned multiplex PCR primer pair combination in the preparation of a multiplex PCR detection kit for Flavobacterium psychrophilum.

[0011] The present invention also provides a multiplex PCR detection kit for Flavobacterium psychrophilum, which includes the above-mentioned multiplex PCR primer pair combination.

[0012] The present invention also provides a multiplex PCR method for detecting Flavobacterium psychrophilum for non-diagnostic purposes, which includes the following steps:

[0013] Genomic DNA of the sample to be detected is extracted;

[0014] Using the genomic DNA as a template, the above-mentioned multiplex PCR primer pair combination is used for multiplex PCR amplification to obtain an amplification product, and electrophoresis detection is carried out;

[0015] It is determined whether the sample to be detected contains Flavobacterium psychrophilum according to the electrophoresis detection result:

[0016] If three electrophoresis bands of 470bp, 610bp and 914bp appear, it is determined that the sample to be detected contains Flavobacterium psychrophilum.

[0017] Furthermore, the reaction system for the multiplex PCR amplification is: 10 μL of 2×Taq Plus PCR Master Mix, 3 μL of a primer pair combination with a volume ratio of 2FP90-F / R:FP1024-F / R:2FP1022-F / R = 3:1:3, 2 μL of DNA template, and ddH 2 O is added to make up to 20 μL; the reaction program for the multiplex PCR amplification is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 90 s, and cyclic amplification for 35 times; extension at 72°C for 10 min.

[0018] The present invention discloses the following technical effects:

[0019] First, the present invention obtained the whole genome sequence of Flavobacterium psychrophilum through high-throughput sequencing technology, compared the whole genome sequence one by one in the NCBI database, found specific sequences that did not match other genomes at all, designed specific primers based on the specific sequences, and detected the specificity of the primers through other strains. Finally, primers with high specificity were screened. After that, a multiplex PCR primer pair combination for Flavobacterium psychrophilum was designed and used for multiplex PCR detection. The results showed that the multiplex PCR primer pair combination designed and screened in the present invention had good amplification effects for detecting the target Flavobacterium psychrophilum, and had significant specificity and high detection sensitivity when used for detecting Flavobacterium psychrophilum. The primer pair combinations 2FP90-F / R, FP1024-F / R, and 2FP1022-F / R could still detect samples with a concentration as low as 12 pg / μL.

[0020] For fish body detection, the kit of the present invention has good application value, provides new technical support for the rapid detection of Flavobacterium psychrophilum, and can contribute to the prevention and control of fish bacterial cold water disease. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 Results of amplifying Flavobacterium psychrophilum with 6 pairs of primers respectively; among them, 1: Marker; 2: primer FP88-F / R; 3: primer FP90-F / R; 4: primer 2FP90-F / R; 5: primer FP995-F / R; 6: primer 2FP1022-F / R; 7: primer FP1024-F / R; 8: primer 27-F / 1492-R;

[0023] Figure 2 Results of primer specificity detection in Example 2; among them, A to F are the gel diagrams obtained by amplifying primers of Vibrio parahaemolyticus, Exiguobacterium acetylicum, Shewanella putrefaciens, Aeromonas hydrophila, Plesiomonas shigelloides, and Bacillus polymyxa respectively; in A to F, 1: Marker; 2: primer FP88-F / R; 3: primer FP90-F / R; 4: primer 2FP90-F / R; 5: primer FP995-F / R; 6: primer 2FP1022-F / R; 7: primer FP1024-F / R; 8: primer 27-F / 1492-R;

[0024] Figure 3Electrophoretogram for screening multiplex PCR primer combinations; among them, 1: Marker; 2 - 12: Combination ① - Combination ;

[0025] Figure 4 Electrophoretogram for optimizing the concentration ratio of multiplex PCR primers; among them, 1: Marker; 2: Combination ①; 3: Combination ⑥; 4: Combination

[0026] Figure 5 Electrophoretogram for the sensitivity of multiplex PCR; among them, A - C respectively correspond to Combination ①, Combination ⑥, and Combination In A, M: DNA Marker; 1 - 15 are respectively the DNA concentrations of Flavobacterium psychrophilum at 120 ng / μL, 24 ng / μL, 12 ng / μL, 2.4 ng / μL, 1.2 ng / μL, 240 pg / μL, 120 pg / μL, 24 pg / μL, 12 pg / μL, 6 pg / μL, 3 pg / μL, 1.5 pg / μL, 0.8 pg / μL, 0.4 pg / μL, 0.2 pg / μL; in B, M: DNA Marker; 1 - 15 are respectively the DNA concentrations of Flavobacterium psychrophilum at 120 ng / μL, 24 ng / μL, 12 ng / μL, 2.4 ng / μL, 1.2 ng / μL, 240 pg / μL, 120 pg / μL, 24 pg / μL, 12 pg / μL, 6 pg / μL, 3 pg / μL, 1.5 pg / μL, 0.8 pg / μL, 0.4 pg / μL, 0.2 pg / μL; in C, M: DNA Marker; 1 - 15 are respectively the DNA concentrations of Flavobacterium psychrophilum at 120 ng / μL, 24 ng / μL, 12 ng / μL, 2.4 ng / μL, 1.2 ng / μL, 240 pg / μL, 120 pg / μL, 24 pg / μL, 12 pg / μL, 6 pg / μL, 3 pg / μL, 1.5 pg / μL, 0.8 pg / μL, 0.4 pg / μL, 0.2 pg / μL. Detailed implementation manners

[0027] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0028] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0031] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0032] Example 1 Primer Specific Design

[0033] The complete genome sequence of Flavobacterium psychrophilum was obtained through high-throughput sequencing technology. The complete genome sequence was uploaded to the NCBI database for pairwise comparison one by one, and 100 - 1000 bp specific sequences that did not match any other sequences in the database were screened out. Primer Premier 5 software was used for primer sequence design, and the NCBI database was used to analyze the primer specificity. Six pairs of primers were designed and named FP88-F / R, FP995-F / R, FP1024-F / R, FP90-F / R, 2FP90-F / R, and 2FP1022-F / R respectively. The primer information and product fragments are shown in Table 1.

[0034] Table 1 Information of 6 pairs of primers and product fragments

[0035]

[0036]

[0037] The genomic DNA of Flavobacterium psychrophilum was amplified by PCR using six pairs of primers respectively, and clear bands of different sizes were obtained, as Figure 1 shown.

[0038] The 20 μL reaction system was used for PCR amplification: 1 μL of each of the upstream and downstream primers (both at a concentration of 5 nmol) was added to the PCR reaction tube, and then 10 μL of 2×Taq Plus PCR Master Mix, 2 μL of DNA template, and 6 μL of ddH 2 O were added respectively.

[0039] The PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 90 s, with 35 cycles of amplification; extension at 72°C for 10 min.

[0040] Example 2 Primer Specificity Detection

[0041] The primers designed in Example 1 were used to perform PCR amplification on the DNA templates of the test strains respectively, and the universal primers 27-F / 1492-R were used as the positive control. The specific operations were as follows:

[0042] (1) Preparation of DNA templates: There were 6 test strains, namely: Vibrio Parahaemolyticus, Exiguobacterium acetylicum, Shewanella putrefaciens, Aeromonas hydrophila, Plesiomonas shigelloides, and Paenibacillus polymyxa.

[0043] The extraction method for the DNA templates of each strain: 1 mL of bacterial liquid was centrifuged at 12000 rpm for 1 min at 4°C in a high-speed refrigerated centrifuge. After discarding the supernatant until 200 μL remained, the bacterial liquid was resuspended, and the bacterial liquid DNA was extracted (TIANGEN DNA extraction kit).

[0044] (2) PCR System

[0045] The 20 μL reaction system was used: 1 μL of each of the upstream and downstream primers (both at a concentration of 5 nmol) was added to the PCR reaction tube, and then 10 μL of 2×Taq Plus PCR Master Mix, 2 μL of DNA template, and 6 μL of ddH 2 O were added respectively.

[0046] PCR amplification conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 90 s, with 35 cycles of amplification; extension at 72°C for 10 min.

[0047] (3) Determination of test results

[0048] Prepare a 1.5% agarose gel plate with TBE electrophoresis buffer, place it in a horizontal electrophoresis tank, and cover the gel surface with the electrophoresis buffer. Take 4 μL of the PCR product and add it to the sample well. After electrophoresis at 120 V voltage and 200 A current for 30 min, place it in a gel imager to view the results.

[0049] The test results of each strain are as Figure 2 shown. The results show that fluorescence appears in lane 8 (primer 27-F / 1492-R) for each pathogenic bacterium, and no fluorescence appears in other lanes, indicating that the primers FP88-F / R, FP90-F / R, 2FP90-F / R, FP995-F / R, 2FP1022-F / R, and FP1024-F / R have no reaction to the above pathogenic bacteria.

[0050] Example 3 uses a primer pair combination for multiplex PCR detection of Flavobacterium psychrophilum

[0051] In order to obtain a primer pair combination for multiplex PCR detection of Flavobacterium psychrophilum, specific gene fragments of Flavobacterium psychrophilum with similar PCR reaction procedures and different PCR product lengths must be screened out as candidate detection target fragments for multiplex PCR.

[0052] Based on the PCR reaction procedure and the length of the PCR product, the following 11 pairs of primer sets can be established, namely:

[0053] Combination ①: FP88-F / R (172 bp), FP995-F / R (325 bp), and FP1024-F / R (610 bp);

[0054] Combination ②: FP88-F / R (172 bp), FP995-F / R (325 bp), and 2FP90-F / R (470 bp);

[0055] Combination ③: FP88-F / R (172 bp), FP995-F / R (325 bp), and 2FP1022-F / R (914 bp);

[0056] Combination ④: FP88-F / R (172 bp), 2FP90-F / R (470 bp), and 2FP1022-F / R (914 bp);

[0057] Combination ⑤: FP88-F / R (172bp), FP1024-F / R (610bp) and 2FP1022-F / R (914bp);

[0058] Combination ⑥: FP90-F / R (197bp), FP995-F / R (325bp) and FP1024-F / R (610bp);

[0059] Combination ⑦: FP90-F / R (197bp), FP995-F / R (325bp) and 2FP90-F / R (470bp);

[0060] Combination ⑧: FP90-F / R (197bp), FP995-F / R (325bp) and 2FP1022-F / R (914bp);

[0061] Combination ⑨: FP90-F / R (197bp), 2FP90-F / R (470bp) and 2FP1022-F / R (914bp);

[0062] Combination ⑩: FP90-F / R (197bp), FP1024-F / R (610bp) and 2FP1022-F / R (914bp);

[0063] Combination 2FP90-F / R (470bp), FP1024-F / R (610bp) and 2FP1022-F / R (914bp).

[0064] Select several groups of multiplex PCR candidate primer pairs with better amplification effects and set up a 20 μL PCR reaction system: 10 μL of 2×Taq Plus PCR Master Mix, 3 μL of total primers (so that the volume ratio of primer pair 1:primer pair 2:primer pair 3 is 1:1:1, and the concentration of each primer is 5 nmol), 2 μL of Flavobacterium psychrophilum DNA template, add ddH 2 O to make up to 20 μL.

[0065] The multiplex PCR reaction program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 90 s, cycle amplification for 35 times; extension at 72°C for 10 min.

[0066] After the PCR reaction, perform agarose gel electrophoresis, and the results are shown in Figure 3 . Determine whether the target fragment of Flavobacterium psychrophilum DNA is contained according to whether clear fluorescent bands appear on the PCR electrophoresis map.

[0067] The results show that only combination ①, combination ⑥ and combination Clear bands can be detected for all three target fragments.

[0068] Optimization of the primer ratio for multiplex PCR of combination ①:

[0069] Set up a 20 μL PCR reaction system: 10 μL of 2×Taq Plus PCR Master Mix, 3 μL of the primer pair combination of FP88-F / R:FP995-F / R:FP1024-F / R = 1:1:3 (volume ratio) (the concentration of each primer is 5 nmol), 2 μL of Flavobacterium psychrophilum DNA template, add ddH 2 O to make up to 20 μL; the multiplex PCR reaction program is: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 90 s, with 35 cycles of amplification; extension at 72 °C for 10 min. The results ( Figure 4 lane 2 in the figure) showed that clear bands appeared for all three target gene fragments, and the brightness difference of the three bands was small.

[0070] Optimization of the primer ratio for multiplex PCR of combination ⑥:

[0071] Set up a 20 μL PCR reaction system: 10 μL of 2×Taq Plus PCR Master Mix, 3 μL of the primer pair combination of FP90-F / R:FP995-F / R:FP1024-F / R = 2:1:2 (volume ratio) (the concentration of each primer is 5 nmol), 2 μL of Flavobacterium psychrophilum DNA template, add ddH 2 O to make up to 20 μL; the multiplex PCR reaction program is: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 90 s, with 35 cycles of amplification; extension at 72 °C for 10 min. The results ( Figure 4 lane 3 in the figure) showed that clear bands appeared for all three target gene fragments, and the brightness difference of the three bands was small.

[0072] For combination Optimization of the primer ratio for multiplex PCR:

[0073] Set up a 20 μL PCR reaction system: 10 μL of 2×Taq Plus PCR Master Mix, 3 μL of the primer pair combination of 2FP90-F / R:FP1024-F / R:2FP1022-F / R = 3:1:3 (the concentration of each primer is 5 nmol), 2 μL of Flavobacterium psychrophilum DNA template, add ddH 2Make up to 20 μL with O; the multiplex PCR reaction program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 90 s, and cycle amplification for 35 times; extension at 72°C for 10 min. Results ( Figure 4 in lane 4 of

[0074] Example 4 Sensitivity Experiment

[0075] To determine the detection sensitivity of the multiplex PCR in Example 3, according to the optimized multiplex PCR reaction conditions and procedures, the extracted DNA of Flavobacterium psychrophilum was serially diluted to form 15 concentration gradients, and the sensitivity of the multiplex PCR was detected.

[0076] The multiplex PCR sensitivity of combination ① (FP88-F / R, FP995-F / R, and FP1024-F / R) was detected, and the results are shown in Figure 5 A, and the results show that when the DNA template amount of Flavobacterium psychrophilum is 3 pg / μL, three target bands can still be amplified simultaneously, and the sizes of the band fragments are consistent with the expectations. It indicates that the sensitivity of the multiplex PCR detection method for Flavobacterium psychrophilum established by the present invention can reach 3 pg / μL.

[0077] The multiplex PCR sensitivity of combination ⑥ (FP90-F / R, FP995-F / R, and FP1024-F / R) was detected, and the results are shown in Figure 5 B, and the results show that when the DNA template amount of Flavobacterium psychrophilum is 6 pg / μL, three target bands can still be amplified simultaneously, and the sizes of the band fragments are consistent with the expectations. It indicates that the sensitivity of the multiplex PCR for Flavobacterium psychrophilum established can reach 6 pg / μL.

[0078] For combination (2FP90-F / R, FP1024-F / R, and 2FP1022-F / R), the multiplex PCR sensitivity was detected, and the results are shown in Figure 5 C, and the results show that when the DNA template amount of Flavobacterium psychrophilum is 12 pg / μL, three target bands can still be amplified simultaneously, and the sizes of the band fragments are consistent with the expectations. It indicates that the sensitivity of the multiplex PCR for Flavobacterium psychrophilum established can reach 12 pg / μL.

[0079] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A multiplex PCR primer pair combination for detecting Flavobacterium psychrophilum, characterized in that: The multiplex PCR primer pair combination consists of primer pairs 2FP90-F / R, FP1024-F / R and 2FP1022-F / R; The FP1024-F / R comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.6; The 2FP1022-F / R comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.11 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.

12. The 2FP90-F / R comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.9 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.

10.

2. Use of the multiplex PCR primer pair combination as claimed in claim 1 in preparing a multiplex PCR detection kit for Flavobacterium psychrophilum.

3. A multiplex PCR detection kit for Flavobacterium psychrophilum, characterized in that: The method comprises the multiplex PCR primer pair combination according to claim 1.

4. A multiplex PCR method for detecting Flavobacterium psychrophilum for non-disease diagnosis purposes, characterized in that: The following steps are involved: Extracting genomic DNA of the sample to be tested; Using the genomic DNA as a template, performing multiplex PCR amplification using the multiplex PCR primer pair combination of claim 1 to obtain amplified products, and performing electrophoresis detection; Determine whether the sample to be tested contains psychrophilic Flavobacterium according to the electrophoresis test result: If three electrophoresis bands of 470 bp, 610 bp and 914 bp appear, it is determined that the sample to be tested contains psychrophilic Flavobacterium.

5. The multiplex PCR method according to claim 4, characterized in that: The reaction system of the multiplex PCR amplification is: 10 μL of 2×Taq Plus PCR Master Mix, 3 μL of a primer pair combination with a volume ratio of 2FP90-F / R:FP1024-F / R:2FP1022-F / R=3:1:3, 2 μL of a DNA template, and ddH2O is added to make up to 20 μL; the reaction procedure of the multiplex PCR amplification is: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, extension at 72°C for 90 seconds, 35 cycles of amplification, and extension at 72°C for 10 minutes.

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