PCR (Polymerase Chain Reaction) primer pair for detecting crucian pathogenic shewanella putrefaciens, product and application of PCR primer pair

Abstract

The invention relates to the technical field of molecular detection of pathogenic microorganisms, in particular to a group of PCR (Polymerase Chain Reaction) primer pairs for detecting pathogenic shewanella putrefaciens of crucian carps, a product and application of the PCR primer pairs. The nucleotide sequences of the PCR primer pair are as shown in SEQ ID NO. 1 and SEQ ID NO. 2. Based on the PCR primer pair, the invention also develops a detection kit which comprises an upstream primer, a downstream primer, 2 * Taq Plus PCR MIX, a standard positive template DNA, a negative control solution and ddH2O. The PCR primer pair provided by the invention fills the blank of detecting the pathogenic shewanella putrefaciens of the crucian carp, has a good amplification effect on detecting the pathogenic shewanella putrefaciens of the target crucian carp, and has remarkable specificity and relatively high detection sensitivity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pathogenic microorganism molecular detection, in particular to a set of PCR primer pairs for detecting Shewanella putrefaciens in Carassius auratus, products and applications thereof. BACKGROUND

[0002] Shewanella putrefaciens is a gram-negative short bacillus widely distributed in nature, and is one of the specific spoilage bacteria causing spoilage of aquatic products. Shewanella putrefaciens has a wide range of survival and can survive and spoil aquatic products in low-temperature environments. Studies have shown that it can spoil stored large yellow croaker and other aquatic products at a temperature below 4℃. The toxic effects of Shewanella putrefaciens not only manifest in the spoilage of stored food, but also cause infection and death of various aquaculture species, including grass carp, co-cultured gibel carp, and half-smooth tongue sole. So far, the diagnosis of pathogenic Shewanella putrefaciens in cultured fish is still performed by the streaking method for bacterial isolation, and the isolated bacteria are then subjected to back-susceptibility test. The biggest disadvantage of this method is that it is time-consuming, and often by the time the identification process is completed, more than half of the cultured fish have already died. Therefore, it is of great practical significance and broad application prospect to develop a more efficient, specific, and rapid molecular diagnostic method.

[0003] Polymerase Chain Reaction (PCR) is an important invention in the field of molecular biology, which can amplify the target gene or a piece of DNA fragment to hundreds of thousands or even millions of times in a very short time, and has the advantages of high specificity and sensitivity, rapidity, and convenience. It has been widely used in the field of molecular biology diagnosis.

[0004] CN103820544B discloses a method for isolating and detecting Shewanella putrefaciens in aquatic products, and CN105132554A discloses a PCR detection kit and method for detecting Shewanella infection in Paa spinosa. However, the detection method of CN103820544B is complex and has a long detection period, and although the method of CN105132554A is convenient, it is aimed at detecting Shewanella putrefaciens in Paa spinosa, not pathogenic Shewanella putrefaciens in Carassius auratus. Therefore, it is necessary to develop a detection method that is simple, rapid, specific, and sensitive for detecting pathogenic Shewanella putrefaciens in Carassius auratus. SUMMARY

[0005] The application aims to provide a set of PCR primer pairs for detecting Shewanella putrefaciens, products and applications thereof, so as to solve the problems in the prior art.

[0006] To achieve the above-mentioned purpose, the application provides the following solutions.

[0007] The application provides a set of PCR primer pairs for detecting Shewanella putrefaciens, wherein the primer pairs comprise a forward primer Q2F with a nucleotide sequence as shown in SEQ ID NO. 1 and a reverse primer Q2R with a nucleotide sequence as shown in SEQ ID NO. 2.

[0008] The application provides an application of the above-mentioned PCR primer pairs in preparing a product for detecting Shewanella putrefaciens.

[0009] Preferably, the detection product comprises a detection reagent, a detection kit and a detection chip.

[0010] The application provides a product for detecting Shewanella putrefaciens, which comprises the above-mentioned PCR primer pairs.

[0011] Preferably, the product for detecting Shewanella putrefaciens further comprises 2x Taq Plus PCR MIX, a standard positive template DNA, a negative control solution and ddH2O.

[0012] Preferably, the detection product comprises a detection reagent, a detection kit and a detection chip.

[0013] The application provides an application of the above-mentioned PCR primer pairs or the above-mentioned product for detecting Shewanella putrefaciens in a non-diagnostic and therapeutic purpose.

[0014] The application provides a method for detecting Shewanella putrefaciens in a non-diagnostic and therapeutic purpose, which comprises the following steps.

[0015] The genomic DNA of the to-be-tested crucian carp is used as a template, and the template is subjected to PCR amplification by using the above-mentioned PCR primer pairs; and whether the to-be-tested crucian carp contains Shewanella putrefaciens is determined according to the PCR amplification result.

[0016] Preferably, if an 84 bp electrophoretic band is amplified by using the PCR primer pair, it is identified that the to-be-tested crucian contains Shewanella putrefaciens.

[0017] Preferably, the PCR amplification system is shown in the following table:

[0018]

[0019] The reaction conditions of the PCR amplification are as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 20 s, 56℃ annealing for 20 s, 72℃ extension for 40 s, cycle amplification for 35 times, 72℃ extension for 8 min, and 4℃ preservation.

[0020] The present application discloses the following technical effects:

[0021] The present application provides a set of PCR primer pairs for detecting Shewanella putrefaciens, wherein the primer pair comprises a forward primer Q2F with a nucleotide sequence as shown in SEQ ID NO. 1 and a reverse primer Q2R with a nucleotide sequence as shown in SEQ ID NO. 2. The PCR primer pair provided by the present application is a specific PCR primer pair designed according to the Shewanella putrefaciens strain XWSfb116S (SUB15551469) gene sequence, and the PCR primer pair has good amplification effect for detecting the target Shewanella putrefaciens, realizes early, rapid and convenient detection of the Shewanella putrefaciens, and provides a basis for preventing and controlling the aquaculture diseases caused by the Shewanella putrefaciens. Therefore, the present application fills the blank of detecting the Shewanella putrefaciens.

[0022] Meanwhile, the present application has the following advantages:

[0023] (1) The Shewanella putrefaciens infection of crucian can be identified in large quantities and rapidly;

[0024] (2) Due to the high sensitivity of the PCR method, the required sample amount is greatly reduced, and the detection cost and workload are also greatly reduced;

[0025] (3) While the Shewanella putrefaciens of the crucian is efficiently and accurately detected, the infection rate and infection intensity can be analyzed according to the band brightness. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0027] Figure 1 PCR detection results of Shewanella putrefaciens of crucian carp; wherein, M: DNA marker; C: negative control; 1: Shewanella putrefaciens;

[0028] Figure 2 is the specific detection result; wherein, M: DNA marker; N: positive control; 1: P. shigelloides; 2: Aeromonas veronii; 3: Pseudomonas fluorescens; 4: Flavobacterium columnare; 5: Aeromonas hydrophila; 6: Escherichia coli;

[0029] Figure 3 PCR detection results of Shewanella putrefaciens of crucian carp; wherein, M: DNA marker; C: negative control; 1: Shewanella putrefaciens; DETAILED DESCRIPTION

[0030] The detailed description set forth below of various example implementations of the application describes and discloses only the particular aspects of the application and is not intended to limit the application to the particular aspects described and / or illustrated herein.

[0031] It should be understood that the terms used herein are merely for describing particular embodiments and are not intended to limit the application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value between any stated value or stated range, as well as any other stated value or intermediate value in the stated range is also included in the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art to the application described and claimed herein.

[0033] Many modifications and variations of this application description of the application can be made without departing from the scope or spirit of the application, which is intended to cover all such modifications and variations as would be within the scope of the claimed application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in its application to the details set forth in the description contained herein or exemplified in the examples.

[0034] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” and the like are open-ended terms that are intended to be synonymous with each other, i.e., to include, but not limited to.

[0035] Example 1 Detection of pathogenic Shewanella putrefaciens

[0036] Bacteria: Pathogenic Shewanella putrefaciens (XWfb1) was provided by Jiangsu Freshwater Fisheries Research Institute, and was also disclosed in the literature “Growth Characteristics, Whole Genome Sequencing and Comparative Genomic Analysis of Shewanella sp. XWfb1 Isolated from Carassius auratus”, and a commitment was made to release it to the public for 20 years from the filing date. The strain of Shewanella putrefaciens was isolated from diseased Carassius auratus and has been confirmed to be able to infect farmed Carassius auratus, causing hemorrhage and body surface ulceration. The strain was resuscitated and cultured for experiments.

[0037] Extraction of template DNA: The pure culture of Shewanella putrefaciens was subjected to bacterial DNA extraction according to the instructions of the bacterial DNA extraction kit produced by Tiangen Biosciences Co., Ltd. ddH2O was used as a negative control.

[0038] Design of primer pairs: Specific PCR primer pairs were designed according to the 16S (SUB15551469) gene sequence of Shewanella putrefaciens strain XWSfb1 isolated from Carassius auratus. The nucleotide sequence of the forward primer Q2F was 5'-TGCCTTCGGGAACTGTGA-3' (SEQ ID NO. 1), and the nucleotide sequence of the reverse primer Q2R was 5'-CGCTCGTTGCGGGACTTA-3' (SEQ ID NO. 2).

[0039] PCR amplification: The extracted DNA of pathogenic Shewanella putrefaciens was used as a template, and SEQ ID NO. 1 and SEQ ID NO. 2 were used as forward and reverse primers for fragment PCR amplification. The reaction system for PCR amplification is shown in Table 1.

[0040] Table 1 Reaction system

[0041]

[0042] PCR amplification reaction conditions: 94°C pre-denaturation for 5 min, 94°C denaturation for 20 s, 56°C annealing for 20 s, 72°C extension for 40 s, 35 cycles of amplification, 72°C extension for 8 min, and 4°C storage.

[0043] Result determination: The result observation adopts the agarose gel electrophoresis method, specifically, 10 μL of PCR product is electrophoresed in 1.5% agarose gel (containing nucleic acid dye 1000x Gene Green, Tiangen), the voltage is 100 V, and the time is 40 min, and the PCR amplification result is observed under the ultraviolet lamp. The determination standard is that the specific 84 bp DNA fragment product appears on the gel, which indicates that the primer pair can identify pathogenic Shewanella putrefaciens of crucian carp.

[0044] Result analysis: The results are shown in Table 1. Figure 1 The results show that the primer pair can specifically obtain an amplification product of 84 bp in size.

[0045] Example 2 Development of a detection kit for pathogenic Shewanella putrefaciens

[0046] A detection kit for pathogenic Shewanella putrefaciens is composed of the following components:

[0047] 0.5 μL of forward primer Q2F (10 mM), 0.5 μL of reverse primer Q2R (10 mM), 12.5 μL of 2x Taq Plus PCR MIX, 1 μL of standard positive template DNA (DNA of pathogenic Shewanella putrefaciens (XWfb1) of crucian carp), 1 μL of negative control solution, and 10.5 μL of ddH2O;

[0048] The negative control solution is ddH2O.

[0049] Example 3 Specificity experiment

[0050] Plesiomonas shigelloides, Aeromonas veronii, Pseudomonas fluorescens, Flavobacterium columnare, and Aeromonas hydrophila, which are five common pathogenic bacteria that infect crucian carp, were provided by Jiangsu Institute of Freshwater Aquatic Products and promised to be released to the outside for 20 years from the filing date.

[0051] According to the method of Example 1, the template DNA of Plesiomonas shigelloides, Aeromonas veronii, Pseudomonas fluorescens, Flavobacterium columnare, and Aeromonas hydrophila, which are five common pathogenic bacteria that infect crucian carp, was extracted, PCR reaction amplification was performed, and the results were determined. Pathogenic Aeromonas veronii (XWfb1) was used as a positive control.

[0052] Result analysis: The amplification results are shown in Table 2. Figure 2 The results show that the PCR detection kit has the expected detection specificity, as the positive control appears a band at 84 bp, while the negative control and the other five bacteria do not appear a band.

[0053] Example 4 Actual sample detection

[0054] The sample of crucian carp: the sample was taken from the mouth of the crucian carp culture pond in two different areas A and B, and the sampling time was April 2024. Among them, the crucian carp in fish pond A showed disease, and the diseased crucian carp showed body surface bleeding and ulceration. Autopsy showed that the intestinal tract was filled with light yellow ascites, with an average weight of 326.2 g. The pond continued to have the same symptoms in early March, and after selecting the severely diseased crucian carp, bacterial isolation, identification and back-sensing, it was finally determined that the infection was caused by Shewanella putrefaciens; The crucian carp in fish pond B showed no disease, and the body surface was healthy, which was healthy crucian carp.

[0055] Extraction of crucian carp template DNA:

[0056] ① Using phenol-chloroform method, aseptically taking the liver of the diseased crucian carp, placing it in a disposable sterile homogenizer, adding 1 mL of genomic lysis solution (the components of the genomic lysis solution are: 0.1 mol / L EDTA, 10 mmol / L Tris-HCl, 15 mmol / L Nacl, 0.4% SDS, 200 μg / ml protease K (add before use)), homogenize and place in a 56°C metal bath for 2 h;

[0057] ② Add 200 μL of chloroform (trichloromethane) to each 1 mL of genomic lysis solution sample, shake vigorously for 20 s, and then place at room temperature for 3 min. Centrifuge at 4°C and 12000 r for 15 min, and then transfer the supernatant to a new Eppendorf tube;

[0058] ③ Add 500 μL of 100% isopropanol to the new upper liquid, and place at room temperature for 10 min. Centrifuge at 4°C and 12000 r for 10 min, and discard the supernatant;

[0059] ④ Add 75% ethanol, invert 10 times, centrifuge at 4°C and 12000 r for 5 min, discard the ethanol, and place at room temperature for 10 min with the cap open. The purified DNA is obtained, which can be detected by agarose electrophoresis after dissolving with DEPC water.

[0060] (2) PCR amplification system

[0061] ① Using the extracted DNA as a template, and SEQ ID NO. 1 and SEQ ID NO. 2 as forward and reverse primer pairs, perform fragment PCR amplification. The reaction system of PCR amplification is shown in Table 1;

[0062] ② The reaction conditions of PCR amplification are as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 20 s, 56°C annealing for 20 s, 72°C extension for 40 s, cycle amplification for 35 times, 72°C extension for 8 min, and 4°C storage.

[0063] Result determination: agarose gel electrophoresis method is adopted, specifically, 10 μL of PCR product is electrophoresed in 1.5% agarose gel (containing nucleic acid dye 1000x Gene Green, Tiangen) for 40 min at 100 V, and the PCR amplification result is observed under ultraviolet lamp. The determination standard is that specific 84 bp DNA fragment product appears on the gel, which indicates that the sample contains pathogenic Shewanella putrefaciens. ddH2O is used as negative control, and pathogenic A. hydrophila (XWfb1) is used as positive control.

[0064] Result analysis: the results are shown in Table 1. Figure 3 As shown in Table 1, the results show that bands appear at 84 bp in the sample of fish pond crucian carp of the sample A and the positive control, while no bands appear in the negative control and the sample of fish pond crucian carp of the sample B, indicating that the detection effect of the primer pair provided in the application meets the expectation. And the result is consistent with the identification result of the industry gold standard, i.e., visceral bacteria separation method. It can be seen that the primer pair provided in the application has very high accuracy.

[0065] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A set of PCR primer pairs for detecting pathogenic Shewanella putrefactive bacteria in crucian carp, characterized in that, The primer pair includes a forward primer Q2F with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer Q2R with a nucleotide sequence as shown in SEQ ID NO.

2.

2. The application of the PCR primer pair according to claim 1 in the preparation of a detection product for pathogenic Shewanella putrefactive bacteria in crucian carp.

3. The application according to claim 2, characterized in that, The testing products include testing reagents, testing kits, and testing chips.

4. A product for detecting pathogenic Shewanella putrefactive bacteria in crucian carp, characterized in that, The product for detecting pathogenic Shewanella putrefactive bacteria in crucian carp includes the PCR primer pair described in claim 1.

5. The Shewanella pathogenic putrefactive bacteria detection product for crucian carp according to claim 4, characterized in that, The product for detecting Shewanella pathogenic putrefactive bacteria in crucian carp also includes 2×Taq Plus PCR MIX, standard positive template DNA, negative control solution, and ddH2O.

6. The Shewanella pathogenic putrefactive bacteria detection product for crucian carp according to claim 4, characterized in that, The testing products include testing reagents, testing kits, and testing chips.

7. The use of the PCR primer pair of claim 1 or the detection product for pathogenic Shewanella putrefaction in crucian carp according to any one of claims 4-6 for non-diagnostic and non-therapeutic purposes.

8. A method for detecting pathogenic *Shewanella putrefactive* in crucian carp for non-diagnostic and non-therapeutic purposes, characterized in that, Includes the following steps: Using the genomic DNA of the crucian carp to be tested as a template, PCR amplification of the template was performed using the PCR primers described in claim 1. The PCR amplification results were used to determine whether the crucian carp to be tested contained Shewanella carpitiformis.

9. The method according to claim 8, characterized in that, If an 84 bp electrophoretic band is amplified using the PCR primer pair, the crucian carp to be tested is identified as containing Shewanella carpitiformis.

10. The method according to claim 8, characterized in that, The PCR amplification system is shown in the table below: The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 20 s, 56℃ annealing for 20 s, 72℃ extension for 40 s, 35 cycles of amplification, 72℃ extension for 8 min, and storage at 4℃.

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