Primer group for simultaneously detecting pseudomonas fluorescens, pseudomonas albicans and aeromonas cainea and application of primer group

Abstract

The invention discloses a primer group for simultaneously detecting pseudomonas fluorescens, pseudomonas albicans and aeromonas cainea and application of the primer group, and the primer group comprises a primer group for detecting the pseudomonas fluorescens, the sequence of which is shown as SEQ ID NO.1-3, a primer group for detecting the pseudomonas albicans, a primer group for detecting the pseudomonas albicans and a primer group for detecting the aeromonas cainea, the sequence of the primer group for detecting the Pseudomonas albicans is shown as SEQ ID NO.4-6; the primer group is used for detecting the Pseudomonas albicans; the sequence of the primer group for detecting the aeromonas caviridis is as shown in SEQ ID NO.7-9. The invention also discloses a kit for detecting the aeromonas caviridis. The 5'ends of the sequences as shown in SEQ ID NO.3, 6 and 9 are connected with fluorophores, and the 3 'ends of the sequences as shown in SEQ ID NO.3, 6 and 9 are connected with quenching groups. The method has the advantages that the pseudomonas fluorescens, the pseudomonas albicans and the aeromonas cainea can be detected at the same time, the accuracy rate is high, the detection speed is high, the cost is low, and the sensitivity is high.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen detection, and particularly to the technology for detecting pathogens by qRT-PCR. Background Art

[0002] The output of aquaculture has been growing rapidly in global food production and occupies an important position in the global economy. Aquaculture plays an important role in providing global nutrition and food security, especially in regions with food shortages. Intensive aquaculture methods have outstanding advantages in aquaculture output, but diseases caused by various pathogenic bacteria and viruses pose major challenges to this aquaculture method. The losses caused by fish diseases account for about 30% or more of the total output.

[0003] The main common infectious diseases in aquaculture are Pseudomonas disease, white skin disease and ulcerative disease caused by Pseudomonas fluorescens, Pseudomonas alcaligenes and Aeromonas caviae. Pseudomonas fluorescens, belonging to the genus Pseudomonas, family Pseudomonadaceae, is a Gram-negative psychrophilic bacterium. The characteristics of Pseudomonas disease are fish ulcers, septicemia, petechial hemorrhage, blackening of the skin, scale shedding, accumulation of ascites and exophthalmos, which can affect a variety of fish, including tilapia, crucian carp and yellow catfish, etc. Pseudomonas alcaligenes can infect fish such as silver carp, bighead carp, grass carp and black carp. After fish are infected, they will get sick and die within only 2 - 3 days, and the mortality rate is as high as 50%. Aeromonas caviae is a Gram-negative rod-shaped bacterium commonly found in aquatic environments and has the potential to cause fish infections, leading to an increase in the morbidity and mortality rates in fish farms. Infections caused by Aeromonas caviae usually manifest as skin and fin hemorrhage, ulcers, abdominal swelling, exophthalmos, eye congestion and fin degeneration.

[0004] The techniques for laboratory detection of the above three pathogens generally rely on culture and non-culture methods. The culture-dependent method is complex and time-consuming, requiring 4 - 7 days for detection, and has limited reproducibility. The non-culture methods mainly rely on molecular detection methods such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Although these methods have good sensitivity and specificity, they also have certain limitations, such as complex operation, easy generation of aerosol contamination and non-specific amplification, etc. Real-time PCR technology has been widely used in pathogen detection in the fields of diagnosis, food safety and environmental monitoring due to its special sensitivity and specificity. However, a multiplex real-time PCR method for simultaneously detecting three pathogenic bacteria, namely Pseudomonas fluorescens, Pseudomonas alcaligenes and Aeromonas caviae, has not been reported yet. If a multiplex real-time PCR detection method with good specificity, high sensitivity and rapidity can be developed for simultaneously detecting the three pathogens of Pseudomonas fluorescens, Pseudomonas alcaligenes and Aeromonas caviae, it is crucial for preventing fish diseases that cause significant economic losses in aquaculture. Summary of the Invention

[0005] The object of the present invention is to provide a primer set for simultaneously detecting Pseudomonas fluorescens, Pseudomonas alboprecipitans and Aeromonas caviae, and its application, so as to solve the problem in the prior art that there is a lack of a technology for quickly and accurately detecting three pathogens, namely Pseudomonas fluorescens, Pseudomonas alboprecipitans and Aeromonas caviae, simultaneously.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A primer set for simultaneously detecting Pseudomonas fluorescens, Pseudomonas alboprecipitans and Aeromonas caviae, comprising:

[0008] The primer set for detecting Pseudomonas fluorescens, the forward primer sequence is as shown in SEQ ID NO.1, the reverse primer sequence is as shown in SEQ ID NO.2, and the TaqMan probe sequence is as shown in SEQ ID NO.3;

[0009] The primer set for detecting Pseudomonas alboprecipitans, the forward primer sequence is as shown in SEQ ID NO.4, the reverse primer sequence is as shown in SEQ ID NO.5, and the TaqMan probe sequence is as shown in SEQ ID NO.6;

[0010] The primer set for detecting Aeromonas caviae, the forward primer sequence is as shown in SEQ ID NO.7, the reverse primer sequence is as shown in SEQ ID NO.8, and the TaqMan probe sequence is as shown in SEQ ID NO.9;

[0011] The 5' ends of the sequences shown in SEQ ID NO.3, 6, and 9 are respectively connected to different fluorescent groups, and the 3' ends are all connected to quenching groups.

[0012] Furthermore, the 5' end of the sequence shown in SEQ ID NO.3 is connected to the FAM group;

[0013] The 5' end of the sequence shown in SEQ ID NO.6 is connected to the CY5 group;

[0014] The 5' end of the sequence shown in SEQ ID NO.9 is connected to the HEX group;

[0015] The 3' ends of the sequences shown in SEQ ID NO.3, 6, and 9 are all connected to the BHQ2 group.

[0016] The present invention also provides an application of the above primer set in the preparation of reagents and kits for detecting Pseudomonas fluorescens, Pseudomonas alboprecipitans and Aeromonas caviae.

[0017] The present invention also provides an application of the above primer set in the preparation of reagents and kits for detecting Pseudomonas fluorescens, Pseudomonas alboprecipitans and Aeromonas caviae, including positive controls and negative controls;

[0018] The positive control is a T-vector clone containing partial fragments of the gyrB gene of Pseudomonas fluorescens, the rpoD gene of Pseudomonas alboprecipitans, and the metG gene of Aeromonas caviae;

[0019] The negative control is ultrapure water.

[0020] The present invention also provides a qRT-PCR detection reaction system containing the primer set described above, and the components include: a qRT-PCR reaction premix, all of the above forward primers and reverse primers, all of the above TaqMan probes, nuclease-free water, and a template.

[0021] The present invention also provides a qRT-PCR detection reaction system containing the primer set described above, and the components include: 10 μL of TaqMan TM FastAdvanced premix, all of the above forward primers and reverse primers, all of the above TaqMan probes, 4.4 μL of nuclease-free water, and 2 μL of template;

[0022] The dosage of all of the above forward primers and reverse primers is 0.5 μL of 10 μM;

[0023] The dosage of all of the above TaqMan probes is 0.2 μL of 10 μM.

[0024] The advantages of the present invention are as follows: it can simultaneously detect Pseudomonas fluorescens, Pseudomonas alboprecipitans, and Aeromonas caviae, with high accuracy, fast detection speed, low cost, and high sensitivity. The minimum detection limit for Pseudomonas fluorescens, Pseudomonas alboprecipitans, and Aeromonas caviae is 10 3 copies / μL. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings described herein are used to provide a further understanding of the present invention, form a part of this application, and do not constitute an improper limitation to the present invention. In the drawings:

[0026] Figure 1 is the specific result diagram of Pseudomonas fluorescens;

[0027] Figure 2 is the specific result diagram of Pseudomonas alboprecipitans;

[0028] Figure 3 is the specific result diagram of Aeromonas caviae;

[0029] Figure 4 is the plasmid sensitivity result diagram of Pseudomonas fluorescens;

[0030] Figure 5 is the plasmid sensitivity result diagram of Pseudomonas alboprecipitans;

[0031] Figure 6 It is the result graph of the plasmid sensitivity of Aeromonas caviae. Specific implementation manners

[0032] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Here, the illustrative embodiments and descriptions of the present invention are used to explain the present invention, but do not limit the present invention.

[0033] 1. Primer sets, reaction systems, and reaction procedures for detecting Pseudomonas fluorescens, Pseudomonas alboprecipitans, and Aeromonas caviae

[0034] Design qRT-PCR primer sets for the gyrB gene of Pseudomonas fluorescens, qRT-PCR primer sets for the rpoD gene of Pseudomonas alboprecipitans, and qRT-PCR primer sets for the metG gene of Aeromonas caviae. The primer set sequences are shown in Table 1.

[0035] Table 1

[0036]

[0037] The qRT-PCR systems for the above three pathogens are as follows:

[0038] 10 μL TaqMan TM Fast Advanced Premix (Thermo Fisher Scientific, catalog number 4444557), all forward primers in Table 1, all reverse primers in Table 1, all TaqMan probes in Table 1, 4.4 μL of nuclease-free water, and 2 μL of template;

[0039] The dosage of all forward primers and reverse primers is 0.5 μL of 10 μM;

[0040] The dosage of all TaqMan probes is 0.2 μL of 10 μM.

[0041] The steps of the qRT-PCR reaction procedure include:

[0042] 50 °C, 5 min; 95 °C, 5 min; 95 °C, 15 s; 60 °C, 30 s; 40 amplification cycles.

[0043] Set up negative control and positive control. The positive control is a T-vector clone containing partial fragments of the gyrB gene of Pseudomonas fluorescens, the rpoD gene of Pseudomonas alboprecipitans, and the metG gene of Aeromonas caviae. The preparation method is as follows: Using the isolated and identified Pseudomonas fluorescens, Pseudomonas alboprecipitans, and Aeromonas caviae as templates respectively, and using the forward primers and reverse primers in Table 1 (PfgyrB-Fw, PfgyrB-Re; PbrpoD-FW, PbrpoD-Re; and AcmetG-Fw, AcmetG-Re) for amplification. The sizes of the obtained gene amplification fragments are 64, 53, and 100 bp respectively. Recover the amplification fragments and connect them to the T-vector by conventional methods, which is the positive control.

[0044] The negative control is ultrapure water.

[0045] 2. Specificity experiment

[0046] Detect Enterobacter sakazakii, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes, Vibrio campbellii, Vibrio mimicus, and Vibrio alginolyticus respectively according to the above qRT-PCR reaction system and reaction procedure, and set up positive control and negative control at the same time. The results are as Figures 1 - 3 shown that the positive control has positive amplification, and the DNA of other samples has no amplification.

[0047] 3. Sensitivity experiment

[0048] Culture the engineered bacteria containing partial fragments of the gyrB gene of Pseudomonas fluorescens, the engineered bacteria containing partial fragments of the rpoD gene of Pseudomonas alboprecipitans, and the engineered bacteria containing partial fragments of the metG gene of Aeromonas caviae respectively. Extract DNA, measure the concentration, and then according to the formula: Y copies / μL = (X g / μL DNA / [DNA length × 660]) × 6.022×10 23 Convert its concentration and copy number, then perform 10-fold serial dilution on it and detect one by one. It can be known that the lowest detection limit for Pseudomonas fluorescens, Pseudomonas alboprecipitans, and Aeromonas caviae is 10 3 copies / μL. The detection results are shown in Figures 4 - 6 (where the concentration value 10E8 represents 10 8 , and the same applies to other concentration values).

[0049] 4. Detection of actual samples

[0050] Detect 55 actual samples such as mandarin fish, carp, snakehead, and Micropterus salmonides simultaneously by the single detection method and the multiplex method in 1 above of the present invention. The detection results are shown in Table 3-5.

[0051] The primers for the single detection method are shown in Table 2 respectively. The reaction system and reaction procedure for single detection are the same as those in the corresponding literature.

[0052] Table 2

[0053]

[0054] Table 3 Comparison of Detection of Fluorescent Pseudomonas aeruginosa in Actual Samples

[0055]

[0056] Table 4 Comparison of Detection of Pseudomonas alboprecipitans in Actual Samples

[0057]

[0058] Table 5 Comparison of Detection of Aeromonas caviae in Actual Samples

[0059]

[0060] It can be seen from the above results that the multiplex primer set detection technology of the present invention has low false negative rate and high accuracy.

[0061] The technical solutions provided by the embodiments of the present invention have been introduced in detail above. Specific examples are used in this article to elaborate on the principles and implementation manners of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention; at the same time, for those of ordinary skill in the art, according to the embodiments of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A primer set for simultaneously detecting Pseudomonas fluorescens, Pseudomonas albicans and Aeromonas caviae, characterized in that: include: A primer set for detecting Pseudomonas fluorescens, wherein the forward primer sequence is shown in SEQ ID NO.1, the reverse primer sequence is shown in SEQ ID NO.2, and the TaqMan probe sequence is shown in SEQ ID NO.3; A primer set for detecting Pseudomonas albicans, wherein the forward primer sequence is shown in SEQ ID NO.4, the reverse primer sequence is shown in SEQ ID NO.5, and the TaqMan probe sequence is shown in SEQ ID NO.6; A primer set for detecting Aeromonas caviae, wherein the forward primer sequence is shown in SEQ ID NO.7, the reverse primer sequence is shown in SEQ ID NO.8, and the TaqMan probe sequence is shown in SEQ ID NO.9; The 5' ends of the sequences shown in SEQ ID NO. 3, 6 and 9 are connected to different fluorescent groups respectively, and the 3' ends are connected to quenching groups.

2. The primer set for simultaneous detection of Pseudomonas fluorescens, Pseudomonas albicans and Aeromonas caviae according to claim 1, characterized in that: The 5' end of the sequence shown in SEQ ID NO.3 is connected to a FAM group; The 5' end of the sequence shown in SEQ ID NO.6 is connected to the CY5 group; The 5' end of the sequence shown in SEQ ID NO.9 is connected to a HEX group; The 3' ends of the sequences shown in SEQ ID NOs. 3, 6 and 9 are all connected to a BHQ2 group.

3. Use of the primer set according to claim 1 or 2 in the preparation of reagents and kits for detecting Pseudomonas fluorescens, Pseudomonas albicans and Aeromonas caviae.

4. Use of the primer set as claimed in claim 3 in the preparation of reagents and kits for detecting Pseudomonas fluorescens, Pseudomonas albicans and Aeromonas caviae, characterized in that: Include positive and negative controls; The positive control is a T vector clone containing a partial fragment of the gyrB gene of Pseudomonas fluorescens, the rpoD gene of Pseudomonas albicans, and the metG gene of Aeromonas caviae; The negative control was ultrapure water.

5. A qRT-PCR detection reaction system comprising the primer set according to claim 1 or 2, characterized in that: Components include: qRT-PCR reaction master mix, all forward and reverse primers, all TaqMan probes, nuclease-free water, and template.

6. A qRT-PCR detection reaction system as claimed in claim 5, characterized in that: Element Includes: 10 μL TaqMan TM FastAdvanced master mix, all forward and reverse primers, all TaqMan probes, 4.4 μL nuclease-free water, and 2 μL template; All forward and reverse primers were used in 0.5 μL of 10 μM; All TaqMan probes were used in 0.2 μL of 10 μM.

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