Primer combination for detecting grouper pathogenic vibrio harveyi and application thereof

Abstract

The invention discloses a primer combination for detecting grouper pathogenic vibrio harveyi and application of the primer combination. The primer combination is a nested PCR primer aiming at the pathogenic vibrio harveyi of groupers, comprises a forward outer primer, a reverse outer primer, a forward inner primer and a reverse inner primer, and can efficiently, sensitively and specifically identify the pathogenic vibrio harveyi in groupers.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pathogen species identification, and particularly relates to a primer combination for detecting the pathogen Vibrio harveyi of grouper and application thereof. BACKGROUND

[0002] Grouper is one of the important marine fish in China, and has high economic value. Vibrio harveyi, as a common marine pathogenic bacteria, often causes infection and disease of grouper, and brings huge economic losses to grouper aquaculture industry. Therefore, rapid and accurate identification of Vibrio harveyi is of great significance for the prevention and control of grouper diseases.

[0003] At present, there are many methods for identifying Vibrio harveyi, among which the conventional PCR technology is widely used due to its relatively simple operation, high specificity and other characteristics. However, the existing conventional PCR identification method still has deficiencies in detection sensitivity and specificity, which is difficult to meet the actual detection needs. For example, when the content of pathogenic bacteria in the sample is low, the conventional PCR may not be able to accurately amplify the target fragment, resulting in missed detection; at the same time, there may be cross-reaction with other similar species, affecting the accuracy of the identification result.

[0004] Nested PCR technology is a nucleic acid amplification technology developed on the basis of conventional PCR. Through two rounds of PCR amplification, using two pairs of inner and outer primers, the specificity and sensitivity of amplification are greatly improved. However, there is no nested PCR primer for Vibrio harveyi used for identification of grouper pathogenic bacteria at present. SUMMARY

[0005] The present application aims to design a set of nested PCR primers for Vibrio harveyi, and to establish an efficient, sensitive and specific identification method for Vibrio harveyi, the pathogen of grouper, by using nested PCR technology, in order to overcome the defects of the existing conventional PCR identification method.

[0006] In the first aspect, the present application provides a primer combination for identifying target genes using nested PCR, which includes an outer primer pair and an inner primer pair, wherein:

[0007] 1) the sequence of the outer primer pair, comprising:

[0008] forward outer primer (VH-outer-F): 5'-GCAACGAAAGCCGTATTCT-3',

[0009] reverse outer primer (VH-outer-R): 5'-AAGCGACGACAACCAAAATA-3';

[0010] 2) the sequence of the inner primer pair, comprising:

[0011] Forward inner primer (VH-inner-F): 5'-TGCATGAGTTCGTTTGGC-3',

[0012] Reverse inner primer (VH-inner-R): 5'-GTGTTGGCTTCGGCTTTT-3'.

[0013] The present application designs nested PCR primers according to the toxR specific gene sequence of Vibrio harveyi, and the nested PCR primers are obtained through intensive research and repeated comparison and screening; wherein the amplification product of the outer primer is 444 bp, and the amplification product of the inner primer is 327 bp.

[0014] In some embodiments, the target gene is a pathogenic microorganism gene, preferably a Vibrio harveyi gene, more preferably a Vibrio harveyi gene in a grouper.

[0015] In a second aspect, the present application provides a nested PCR amplification method, comprising the step of using the primer combination of the first aspect.

[0016] In some embodiments, the nested PCR amplification method comprises the following steps:

[0017] The primer combination of the first aspect is used to perform PCR amplification on the sample to be tested;

[0018] In some embodiments, the PCR amplification can be one round of PCR amplification or two rounds of PCR amplification.

[0019] In some embodiments, the method comprises the following steps:

[0020] Step one: extract total DNA from the sample to be tested, and use the outer primer pair to perform the first round of nested PCR amplification with the total DNA as the template;

[0021] Step two: take the first round of nested PCR amplification product as the template, and use the inner primer pair to perform the second round of nested PCR amplification to obtain the amplification target product;

[0022] Step three: perform agarose gel electrophoresis on the amplification target product, and identify the target gene according to the electrophoresis result.

[0023] In some embodiments, the target gene is a pathogenic microorganism gene, preferably a Vibrio harveyi gene, more preferably a Vibrio harveyi gene in a grouper;

[0024] In some embodiments, the sample to be tested is a sample of body fluid of a grouper, preferably a blood sample and / or a tissue sample, more preferably a liver and / or spleen tissue sample of a grouper;

[0025] In some embodiments, the conditions for the first round of nested PCR amplification are:

[0026] 94-95°C pre-denaturation for 5 minutes (e.g., can be 94°C, 94.1°C, 94.2°C, 94.3°C, 94.4°C, 94.5°C, 94.6°C, 94.7°C, 94.8°C, 94.9°C, or all values within the range due to page limitation not listed one by one); 94-96°C denaturation for 30 seconds (e.g., can be 94°C, 94.1°C, 94.2°C, 94.3°C, 94.4°C, 94.5°C, 94.6°C, 94.7°C, 94.8°C, 94.9°C, 95°C, 95.1°C, 95.2°C, 95.3°C, 95.4°C, 95.5°C, 95.6°C, 95.7°C, 95.8°C, 95.9°C, 96°C, or all values within the range due to page limitation not listed one by one), 55-57°C annealing for 30 seconds (e.g., can be 55°C, 55.1°C, 55.2°C, 55.3°C, 55.4°C, 55.5°C, 55.6°C, 55.7°C, 55.8°C, 55.9°C, 56°C, 56.1°C, 56.2°C, 56.3°C, 56.4°C, 56.5°C, 56.6°C, 56.7°C, 56.8°C, 56.9°C, 57°C, or all values within the range due to page limitation not listed one by one), 72-74°C extension for 30 seconds (e.g., can be 72°C, 72.1°C, 72.2°C, 72.3°C, 72.4°C, 72.5°C, 72.6°C, 72.7°C, 72.8°C, 72.9°C, 73°C, 73.1°C, 73.2°C, 73.3°C, 73.4°C, 73.5°C, 73.6°C, 73.7°C, 73.8°C, 73.9°C, 74°C, or all values within the range due to page limitation not listed one by one), for 30-35 cycles; 72-74°C re-extension for 10 minutes (e.g., can be 72°C, 72.1°C, 72.2°C, 72.3°C, 72.4°C, 72.5°C, 72.6°C, 72.7°C, 72.8°C, 72.9°C, 73°C, 73.1°C, 73.2°C, 73.3°C, 73.4°C, 73.5°C, 73.6°C, 73.7°C, 73.8°C, 73.9°C, 74°C, or all values within the range due to page limitation not listed one by one), and finally 3-6°C incubation (e.g., can be 3°C, 3.2°C, 3.4°C, 3.6°C, 3.8°C, 4°C, 4.2°C, 4.4°C, 4.6°C, 4.8°C, 5°C, 5.2°C, 5.4°C, 5.6°C, 5.8°C, 6°C, or all values within the range due to page limitation not listed one by one);

[0027] In some embodiments, the conditions for the second round of nested PCR amplification are:

[0028] 94-95°C pre-denaturation for 5 minutes (for example, it can be 94°C, 94.1°C, 94.2°C, 94.3°C, 94.4°C, 94.5°C, 94.6°C, 94.7°C, 94.8°C, 94.9°C, or all values within the range, due to the limitation of the length, not listed one by one); 94-96°C denaturation for 30 seconds (for example, it can be 94°C, 94.1°C, 94.2°C, 94.3°C, 94.4°C, 94.5°C, 94.6°C, 94.7°C, 94.8°C, 94.9°C, 95°C, 95.1°C, 95.2°C, 95.3°C, 95.4°C, 95.5°C, 95.6°C, 95.7°C, 95.8°C, 95.9°C, 96°C, or all values within the range, due to the limitation of the length, not listed one by one), 55-57°C annealing for 30 seconds (for example, it can be 55°C, 55.1°C, 55.2°C, 55.3°C, 55.4°C, 55.5°C, 55.6°C, 55.7°C, 55.8°C, 55.9°C, 56°C, 56.1°C, 56.2°C, 56.3°C, 56.4°C, 56.5°C, 56.6°C, 56.7°C, 56.8°C, 56.9°C, 57°C, or all values within the range, due to the limitation of the length, not listed one by one), 72-74°C extension for 30 seconds (for example, it can be 72°C, 72.1°C, 72.2°C, 72.3°C, 72.4°C, 72.5°C, 72.6°C, 72.7°C, 72.8°C, 72.9°C, 73°C, 73.1°C, 73.2°C, 73.3°C, 73.4°C, 73.5°C, 73.6°C, 73.7°C, 73.8°C, 73.9°C, 74°C, or all values within the range, due to the limitation of the length, not listed one by one), 30-35 cycles; 72-74°C re-extension for 10 minutes (for example, it can be 72°C, 72.1°C, 72.2°C, 72.3°C, 72.4°C, 72.5°C, 72.6°C, 72.7°C, 72.8°C, 72.9°C, 73°C, 73.1°C, 73.2°C, 73.3°C, 73.4°C, 73.5°C, 73.6°C, 73.7°C, 73.8°C, 73.9°C, 74°C, or all values within the range, due to the limitation of the length, not listed one by one), and finally 3-6°C incubation (for example, it can be 3°C, 3.2°C, 3.4°C, 3.6°C, 3.8°C, 4°C, 4.2°C, 4.4°C, 4.6°C, 4.8°C, 5°C, 5.2°C, 5.4°C, 5.6°C, 5.8°C, 6°C, or all values within the range, due to the limitation of the length, not listed one by one).

[0029] In a third aspect, the present application provides a reagent kit for identifying the pathogen of Epinephelus, which comprises at least the primer combination of the first aspect.

[0030] In a fourth aspect, the present application provides a sequencing reagent kit, comprising the primer combination of the first aspect, or the kit of the third aspect.

[0031] In a fifth aspect, the present application provides the primer combination of the first aspect, the method of the second aspect, the kit of the third aspect, or the sequencing reagent kit of the fourth aspect for use in any of a1) - a2):

[0032] a1) detecting a pathogenic microorganism for non-diagnostic purposes;

[0033] a2) preparing a product for detecting a pathogenic microorganism.

[0034] In some embodiments, the pathogenic microorganism in a1) - a2) is a pathogenic microorganism in a grouper;

[0035] In some embodiments, the pathogenic microorganism in a grouper is Vibrio harveyi in a grouper.

[0036] Compared with the prior art, the primer combination and its application provided by the present application have at least the following beneficial effects:

[0037] 1. Higher sensitivity: The nested PCR primers are designed according to the specific gene sequence of Vibrio harveyi, and are obtained through intensive research and repeated comparison and screening. The two-round PCR amplification using the primers can significantly improve the detection sensitivity of Vibrio harveyi, and the pathogenic bacteria can be accurately detected even in the case of low content of pathogenic bacteria in the sample.

[0038] 2. Stronger specificity: The nested PCR primers designed in the present application are directed to the specific gene sequence of Vibrio harveyi, and the synergistic effect of the outer primer and the inner primer greatly reduces the cross-reaction with other bacteria, and improves the specificity of the identification result.

[0039] 3. Simple operation and verifiable result: The method of the present application has relatively simple operation steps and short amplification time, and can quickly realize the identification of Vibrio harveyi, meet the demand of rapid detection in actual detection work, and the PCR product can be further sequenced to verify the result to ensure the reliability of the result. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiment or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to the structures shown in these drawings without creative labor.

[0041] Figure 1 Figure of electrophoresis result obtained by nested PCR method in Example 1 and Comparative Examples 1-2.

[0042] The object, features and advantages of the present application will be further illustrated in conjunction with the embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0043] In view of the deficiencies in the prior art, the present inventors have made intensive research and designed a set of nested PCR primers for Vibrio harveyi, and established a highly efficient, sensitive and specific identification method for the pathogenic Vibrio harveyi of grouper by using the nested PCR technology, so as to overcome the defects of the conventional PCR identification method.

[0044] To this end, the present application provides the following technical solutions:

[0045] In a first aspect, the present application provides a primer combination for identifying a target gene by using nested PCR, which comprises an outer primer pair and an inner primer pair, wherein:

[0046] The sequence of the outer primer pair comprises:

[0047] Forward outer primer (VH-outer-F): 5'-GCAACGAAAGCCGTATTCT-3',

[0048] Reverse outer primer (VH-outer-R): 5'-AAGCGACGACAACCAAAATA-3';

[0049] The sequence of the inner primer pair comprises:

[0050] Forward inner primer (VH-inner-F): 5'-TGCATGAGTTCGTTTGGC-3',

[0051] Reverse inner primer (VH-inner-R): 5'-GTGTTGGCTTCGGCTTTT-3'.

[0052] The present application designs the nested PCR primers according to the specific gene sequence of the toxR gene of Vibrio harveyi, and then obtains the above-mentioned nested PCR primers through intensive research and repeated comparison and screening; wherein the amplification product of the outer primer is 444bp, and the amplification product of the inner primer is 327bp.

[0053] In some embodiments, the target gene is a pathogenic microorganism gene, preferably a Vibrio harveyi gene, and more preferably a Vibrio harveyi gene in the body of a grouper.

[0054] In a second aspect, the present application provides a nested PCR amplification method, comprising the step of using the primer combination of the first aspect.

[0055] In some embodiments, the nested PCR amplification method comprises the following steps:

[0056] The sample to be tested is subjected to PCR amplification using the primer combination of the first aspect;

[0057] In some embodiments, the PCR amplification can be one round of PCR amplification or two rounds of PCR amplification.

[0058] In some embodiments, the method comprises the following steps:

[0059] Step 1: Extract total DNA from the sample to be tested, and use the outer primer pair to perform the first round of nested PCR amplification with the total DNA as the template;

[0060] Step 2: Take the first round of nested PCR amplification product as the template, and use the inner primer pair to perform the second round of nested PCR amplification to obtain the target amplification product;

[0061] Step 3: Perform agarose gel electrophoresis on the target amplification product, and identify the target gene according to the electrophoresis result.

[0062] In some embodiments, the target gene is a pathogenic microorganism gene, preferably a Vibrio harveyi gene, and more preferably a Vibrio harveyi gene in the body of a grouper;

[0063] In some embodiments, the sample to be tested is a body fluid sample of a grouper, preferably a blood sample and / or a tissue sample, and more preferably a liver and / or spleen tissue sample of a grouper;

[0064] In some embodiments, the conditions for the first round of nested PCR amplification are as follows:

[0065] 94-95°C pre-denaturation for 5 minutes (for example, it can be 94°C, 94.1°C, 94.2°C, 94.3°C, 94.4°C, 94.5°C, 94.6°C, 94.7°C, 94.8°C, 94.9°C, or all values within the range due to the limitation of the length of the paper, it is not listed one by one); 94-96°C denaturation for 30 seconds (for example, it can be 94°C, 94.1°C, 94.2°C, 94.3°C, 94.4°C, 94.5°C, 94.6°C, 94.7°C, 94.8°C, 94.9°C, 95°C, 95.1°C, 95.2°C, 95.3°C, 95.4°C, 95.5°C, 95.6°C, 95.7°C, 95.8°C, 95.9°C, 96°C, or all values within the range due to the limitation of the length of the paper, it is not listed one by one), 55-57°C annealing for 30 seconds (for example, it can be 55°C, 55.1°C, 55.2°C, 55.3°C, 55.4°C, 55.5°C, 55.6°C, 55.7°C, 55.8°C, 55.9°C, 56°C, 56.1°C, 56.2°C, 56.3°C, 56.4°C, 56.5°C, 56.6°C, 56.7°C, 56.8°C, 56.9°C, 57°C, or all values within the range due to the limitation of the length of the paper, it is not listed one by one), 72-74°C extension for 30 seconds (for example, it can be 72°C, 72.1°C, 72.2°C, 72.3°C, 72.4°C, 72.5°C, 72.6°C, 72.7°C, 72.8°C, 72.9°C, 73°C, 73.1°C, 73.2°C, 73.3°C, 73.4°C, 73.5°C, 73.6°C, 73.7°C, 73.8°C, 73.9°C, 74°C, or all values within the range due to the limitation of the length of the paper, it is not listed one by one), 30-35 cycles; 72-74°C re-extension for 10 minutes (for example, it can be 72°C, 72.1°C, 72.2°C, 72.3°C, 72.4°C, 72.5°C, 72.6°C, 72.7°C, 72.8°C, 72.9°C, 73°C, 73.1°C, 73.2°C, 73.3°C, 73.4°C, 73.5°C, 73.6°C, 73.7°C, 73.8°C, 73.9°C, 74°C, or all values within the range due to the limitation of the length of the paper, it is not listed one by one), and finally 3-6°C incubation (for example, it can be 3°C, 3.2°C, 3.4°C, 3.6°C, 3.8°C, 4°C, 4.2°C, 4.4°C, 4.6°C, 4.8°C, 5°C, 5.2°C, 5.4°C, 5.6°C, 5.8°C, 6°C, or all values within the range due to the limitation of the length of the paper, it is not listed one by one);

[0066] In some embodiments, the conditions for the second round of nested PCR amplification are:

[0067] 94-95°C pre-denaturation for 5 minutes (for example, it can be 94°C, 94.1°C, 94.2°C, 94.3°C, 94.4°C, 94.5°C, 94.6°C, 94.7°C, 94.8°C, 94.9°C, or all values within the range, due to the limitation of the length, not listed one by one); 94-96°C denaturation for 30 seconds (for example, it can be 94°C, 94.1°C, 94.2°C, 94.3°C, 94.4°C, 94.5°C, 94.6°C, 94.7°C, 94.8°C, 94.9°C, 95°C, 95.1°C, 95.2°C, 95.3°C, 95.4°C, 95.5°C, 95.6°C, 95.7°C, 95.8°C, 95.9°C, 96°C, or all values within the range, due to the limitation of the length, not listed one by one), 55-57°C annealing for 30 seconds (for example, it can be 55°C, 55.1°C, 55.2°C, 55.3°C, 55.4°C, 55.5°C, 55.6°C, 55.7°C, 55.8°C, 55.9°C, 56°C, 56.1°C, 56.2°C, 56.3°C, 56.4°C, 56.5°C, 56.6°C, 56.7°C, 56.8°C, 56.9°C, 57°C, or all values within the range, due to the limitation of the length, not listed one by one), 72-74°C extension for 30 seconds (for example, it can be 72°C, 72.1°C, 72.2°C, 72.3°C, 72.4°C, 72.5°C, 72.6°C, 72.7°C, 72.8°C, 72.9°C, 73°C, 73.1°C, 73.2°C, 73.3°C, 73.4°C, 73.5°C, 73.6°C, 73.7°C, 73.8°C, 73.9°C, 74°C, or all values within the range, due to the limitation of the length, not listed one by one), 30-35 cycles; 72-74°C re-extension for 10 minutes (for example, it can be 72°C, 72.1°C, 72.2°C, 72.3°C, 72.4°C, 72.5°C, 72.6°C, 72.7°C, 72.8°C, 72.9°C, 73°C, 73.1°C, 73.2°C, 73.3°C, 73.4°C, 73.5°C, 73.6°C, 73.7°C, 73.8°C, 73.9°C, 74°C, or all values within the range, due to the limitation of the length, not listed one by one), and finally 3-6°C incubation (for example, it can be 3°C, 3.2°C, 3.4°C, 3.6°C, 3.8°C, 4°C, 4.2°C, 4.4°C, 4.6°C, 4.8°C, 5°C, 5.2°C, 5.4°C, 5.6°C, 5.8°C, 6°C, or all values within the range, due to the limitation of the length, not listed one by one).

[0068] In a third aspect, the present application provides a reagent kit for identifying the pathogen of Epinephelus, which comprises at least the primer combination of the first aspect.

[0069] In a fourth aspect, the present application provides a sequencing reagent kit comprising the primer combination of the first aspect, or the kit of the third aspect.

[0070] In a fifth aspect, the present application provides the primer combination of the first aspect, the method of the second aspect, the kit of the third aspect, or the sequencing reagent kit of the fourth aspect for use in any of a1) - a2):

[0071] a1) detecting a pathogenic microorganism for non-diagnostic purposes;

[0072] a2) preparing a product for detecting a pathogenic microorganism.

[0073] In some embodiments, the pathogenic microorganism in a1) - a2) is a pathogenic microorganism in a grouper;

[0074] In some embodiments, the pathogenic microorganism in a grouper is Vibrio harveyi in a grouper.

[0075] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0076] It should be noted that all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present application are only used to explain the relative positional relationship, movement condition, etc. between components in a certain posture (as shown in the drawings), and if the certain posture changes, the directional indications also change accordingly.

[0077] In addition, the description of "first", "second", etc. in the present application is only for the purpose of description, and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" can explicitly or implicitly include at least one of the features. In addition, the technical solutions of various embodiments can be combined with each other, but it must be based on the fact that a person of ordinary skill in the art can realize it, and when the combination of technical solutions contradicts each other or cannot be realized, it should be considered that the combination of technical solutions does not exist, and is also not within the scope of protection required by the present application.

[0078] Example 1

[0079] (1) Primer synthesis: according to the designed outer primer and inner primer sequences, the primers were synthesized by a certain biological company. After purification, the synthesized primers were dissolved in sterile deionized water to prepare a stock solution, which was stored at -20℃ for standby use.

[0080] (2) Template DNA extraction

[0081] The tissue sample of Epinephelus was taken, and the total DNA was extracted by using the commercial DNA extraction kit [DNeasy Blood & Tissue Kit (brand: QIAGEN, item number: 69506)] according to the operation steps of the kit instruction. The extracted DNA was detected by agarose gel electrophoresis and ultraviolet spectrophotometer for purity and concentration, and the qualified DNA sample was stored at -20℃ for standby use.

[0082] (3) First round of PCR amplification

[0083] ① The reaction system (25 μL) was as follows: 10 × PCR Buffer 2.5 μL, dNTP Mixture (2.5 mmol / L) 2 μL, forward outer primer (10 μmol / L) 1 μL, reverse outer primer (10 μmol / L) 1 μL, Taq DNA polymerase (5 U / μL) 0.2 μL, template DNA 2 μL, and sterile deionized water was added to 25 μL.

[0084] ② The reaction conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, a total of 30 cycles; 72℃ final extension for 10 min; 4℃ storage.

[0085] (4) Second round of PCR amplification

[0086] ① The reaction system (25 μL) was as follows: 10 × PCR Buffer 2.5 μL, dNTP Mixture (2.5 mmol / L) 2 μL, forward inner primer (10 μmol / L) 1 μL, reverse inner primer (10 μmol / L) 1 μL, Taq DNA polymerase (5 U / μL) 0.2 μL, first round of PCR amplification product 1 μL, and sterile deionized water was added to 25 μL.

[0087] ② The reaction conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, a total of 35 cycles; 72℃ final extension for 10 min; 4℃ storage.

[0088] (5) Result identification

[0089] Take 5 μL of the second round of PCR amplification product, mix with an appropriate amount of loading buffer, and load into a 1.5% agarose gel for electrophoresis, with a DNA molecular weight standard. After electrophoresis, observe the results of the low copy concentration Vibrio harveyi sample under the ultraviolet gel imaging system. If a specific band of the expected size appears, it is determined to be positive, indicating that the primer combination method can detect low copy concentration Vibrio harveyi. If no specific band appears, it is determined to be negative, indicating that low copy concentration Vibrio harveyi cannot be detected.

[0090] Comparative Example 1

[0091] Select samples known to contain Vibrio harveyi (positive control), samples of Epinephelus fuscoguttatus confirmed not to be infected with Vibrio harveyi, and other common marine pathogenic bacteria samples, and identify them using the nested PCR method of Example 1.

[0092] Comparative Example 2

[0093] Identify low copy concentration Vibrio samples (10 2 copies / mL) using the conventional PCR method. The specific steps are as follows: prepare 25 μL of the reaction system on ice, containing 10×buffer 2.5 μL, dNTPs 2 μL, upper and lower primers (of which the upper F primer is 5'-CTCAAGCGATCTCTACACTGC-3', and the lower R primer is 5'-CTGTGGTAGCTTCAGCAAATT-3') each 1 μL, low copy concentration Vibrio template (10 2 copies / mL) 2 μL, Taq enzyme 0.25 μL, and water 16.25 μL. The PCR program is set as follows: 94℃ pre-denaturation for 5 min, followed by 35 cycles of 94℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 30 s, and finally 72℃ extension for 10 min and storage at 4℃; the product is observed for specific bands by 1.5% agarose gel electrophoresis (100 V, 30 min), and the theoretical amplification product size is 213 bp.

[0094] Result analysis

[0095] Figure 1 The electrophoresis result graph obtained by the nested PCR method in Example 1 and Comparative Example 1. Among them, M is a DNA molecular weight standard, band 1 is the positive control (a known Vibrio harveyi infected sample) in Comparative Example 1, band 2 is the low copy concentration Vibrio sample (10 2 copies / mL) in Example 1, and band 3 is the low copy concentration Vibrio sample (10 2Band 4 is the negative control in Comparative Example 1, and Band 5 is the other pathogenic bacteria sample in Comparative Example 1.

[0096] The agarose gel electrophoresis result shows that Band 1 (positive control) and Band 2 (low copy concentration sample) both have obvious amplification bands, the conventional amplification (Band 3), the Epinephelus sample confirmed not to be infected with Vibrio harveyi (negative control, Band 4) and the other common marine pathogenic bacteria sample (Band 5) do not have specific bands, indicating that the method of the present application has good specificity and accuracy, and the low copy concentration sample in Band 2 confirms the presence of Vibrio harveyi.

[0097] The above results can show that the nested PCR method of the present application can detect Vibrio harveyi at a lower concentration (as low as 10 2 copies / mL) and has no cross-reaction with other pathogenic bacteria, and the identification effect is obviously better than that of the conventional PCR method.

[0098] The above only describes the preferred embodiments of the present application, and does not limit the patent scope of the present application, and any equivalent structural transformation made under the inventive concept of the present application, and directly / indirectly applied in other related technical fields are included in the patent protection scope of the present application.

Claims

1. A primer combination for identifying a target gene using nested PCR, characterized in that, The primer set includes an outer primer pair and an inner primer pair, wherein: 1) Outer primer pair sequences, including: Forward outer primer: 5'-GCAACGAAAGCCGTATTCT-3', Reverse outer primer: 5'-AAGCGACGACAACCAAAATA-3'; 2) Inner primer pair sequences, including: Forward inner primer: 5'-TGCATGAGTTCGTTTGGC-3', Reverse inner primer: 5'-GTGTTGGCTTCGGCTTTT-3'.

2. The primer combination according to claim 1, characterized in that, The target gene is a pathogenic microbial gene, preferably a Vibrio harveyi gene, and more preferably a Vibrio harveyi gene from grouper.

3. A nested PCR amplification method, characterized in that, The step includes using the primer combination as described in claim 1 or 2.

4. The method according to claim 3, characterized in that, The nested PCR amplification method includes the following steps: PCR amplification of the sample to be tested was performed using the primer combination described in claim 1 or 2. In some implementations, the PCR amplification may be a single round of PCR amplification or two rounds of PCR amplification.

5. The method according to claim 4, characterized in that, The method includes the following steps: Step 1: Extract total DNA from the sample to be tested, and use the total DNA as a template to perform the first round of nested PCR amplification using the outer primer pair; Step 2: Using the first round of nested PCR amplification products as templates, perform a second round of nested PCR amplification using inner primer pairs to obtain the target amplification product; Step 3: Perform agarose gel electrophoresis on the amplified target product and identify the target gene based on the electrophoresis results.

6. The method according to claim 3, characterized in that, The target gene is a pathogenic microbial gene, preferably a Vibrio harveyi gene, and more preferably a Vibrio harveyi gene from grouper. In some embodiments, the sample to be tested is a grouper body fluid sample, preferably a blood sample and / or tissue sample, and more preferably a grouper liver and / or spleen tissue sample. In some implementations, the conditions for the first round of nested PCR amplification are as follows: Pre-denaturation at 94-95℃ for 5 minutes; denaturation at 94-96℃ for 30 seconds; annealing at 55-57℃ for 30 seconds; extension at 72-74℃ for 30 seconds; repeat 30-35 times; extend at 72-74℃ for another 10 minutes; and finally hold at 3-6℃. In some implementations, the conditions for the second round of nested PCR amplification are as follows: Pre-denaturation at 94-95℃ for 5 minutes; denaturation at 94-96℃ for 30 seconds, annealing at 55-57℃ for 30 seconds, extension at 72-74℃ for 30 seconds, repeat 30-35 times; extension at 72-74℃ for another 10 minutes, and finally heat treatment at 3-6℃.

7. A kit for identifying pathogens in grouper, characterized in that, The kit includes at least the primer combination as described in claim 1 or 2.

8. A sequencing reagent kit, characterized in that, Includes the primer combination as described in claim 1 or 2, or the kit as described in claim 7.

9. The use of the primer combination of claim 1 or 2, the method of any one of claims 3-6, the kit of claim 7, or the sequencing reagent kit of claim 8 in any one of a1)-a2): a1) Detection of pathogenic microorganisms for non-diagnostic purposes; a2) Prepare products for detecting pathogenic microorganisms.

10. The application according to claim 9, characterized in that, The pathogenic microorganisms mentioned in a1)-a2) are pathogenic microorganisms in the grouper; In some embodiments, the pathogenic microorganism in the grouper is Vibrio harveyi, which is found in the grouper.

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