A method for detecting pathogenic microorganisms in ship ballast water and application thereof

Abstract

The application discloses a kind of ship ballast water pathogenic microorganism detection method and application thereof, belong to biotechnology field.The application is distinguished and identified to the main pathogenic microorganism in ship ballast water, and multiple qPCR detection primer probe is designed according to its conservation area, constructs multiple detection system, can realize the detection and identification of multiple ship ballast water pathogenic microorganism by once reaction, the detection system has good specificity and detection sensitivity, and the minimum can reach 2x10 ‑1 Copy / μl, greatly reduces the detection time length of conventional PCR, realizes efficient and rapid identification, has greater application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for detecting pathogenic microorganisms in ship ballast water and application thereof. BACKGROUND

[0002] Ship ballast water refers to water and suspended matter loaded on a ship to control the ship's heeling, pitching, draft, stability or stress. Ship ballast water contains a large number of organisms, including plankton, microorganisms, bacteria and even small fish, as well as eggs, larvae or spores of various species. Some of these organisms die during the voyage with the ship because they cannot adapt to changes in temperature, salinity, etc., but some can survive and eventually be discharged into a new environment with the ship ballast water. This results in the transfer of a water area's biological or diverse biological groups to another geographically isolated water area. If these organisms can grow, reproduce, establish populations in natural or semi-natural ecosystems or habitats due to lack of natural enemies or other reasons, they may threaten the ecosystem structure and species diversity of these bays, estuaries or inland waters, become invasive species, and the ballast water can also spread harmful parasites and pathogens, and even cause local species extinction.

[0003] With the continuous expansion of the international shipping industry and the increasing awareness of marine environmental protection, the problem of invasive marine organisms caused by the discharge of ship ballast water has attracted widespread attention from all sectors of society. Recognizing the cross-regional transfer of harmful aquatic organisms and pathogens in ship ballast water has had an undeniable impact on the global marine ecological environment, and the Global Environment Fund (GEF) has listed it as one of the four threats to the ocean. In summary, it is particularly important to establish a detection and identification method for pathogenic microorganisms in ship ballast water. SUMMARY

[0004] In view of the technical problems existing in the prior art, the present application provides a method for detecting pathogenic microorganisms in ship ballast water and application thereof. The present application distinguishes and identifies the main pathogenic microorganisms in ship ballast water, and designs multiple qPCR detection primer probes according to the conserved regions to construct a multiple detection system, which can realize the detection and identification of multiple pathogenic microorganisms in ship ballast water through one reaction, greatly reducing the detection time of conventional PCR, achieving efficient and rapid identification, and having great application prospect.

[0005] The primary object of the present application is to provide a kit for rapidly detecting pathogenic microorganisms in ship ballast water, which comprises multiple qPCR reagents for Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Streptococcus faecalis and Vibrio alginolyticus.

[0006] Preferably, the multiple qPCR reagents comprise specific amplification primers and probes.

[0007] Preferably, the specific amplification primer sequences are shown in seq id no. 1-10, in order.

[0008] Preferably, the specific amplification probe sequences are shown in seq id no. 11-15, in order.

[0009] Preferably, the multiplex qPCR reagent further comprises multiplex PCR 5x master mix, deionized water and / or template DNA.

[0010] Another object of the present application is to provide a multiplex qPCR reagent for rapid detection of pathogenic microorganisms in ship ballast water, which comprises specific amplification primers and probes for simultaneously detecting Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Streptococcus faecalis and Vibrio alginolyticus.

[0011] Preferably, the specific amplification primer sequences are shown in seq id no. 1-10, in order.

[0012] Preferably, the specific amplification probe sequences are shown in seq id no. 11-15, in order.

[0013] Preferably, the multiplex qPCR reagent further comprises multiplex PCR 5x master mix, deionized water and / or template DNA.

[0014] Another object of the present application is to provide a multiplex qPCR reagent for rapid detection of pathogenic microorganisms in ship ballast water, which comprises specific amplification primers and probes for simultaneously detecting Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Streptococcus faecalis and Vibrio alginolyticus.

[0015] Preferably, the specific amplification primer sequences are shown in seq id no. 1-10, in order.

[0016] Preferably, the specific amplification probe sequences are shown in seq id no. 11-15, in order.

[0017] Preferably, the multiplex qPCR reagent further comprises multiplex PCR 5x master mix, deionized water and / or template DNA.

[0018] Another aspect of the present application is to provide the use of the kit or reagent in the preparation of a product for detecting pathogenic microorganisms in ship ballast water.

[0019] Preferably, the use comprises a detection method comprising the following steps:

[0020] 1) Extracting DNA of the sample to be tested;

[0021] 2) Using the multiplex qPCR reagent or the kit to perform a multiplex qPCR reaction with the DNA as a template;

[0022] 3) Recording the fluorescence signal in the product by using an enzyme label meter.

[0023] Preferably, the total reaction system of the multiplex qPCR in step 2) is 40 μl, including multiplex PCR 5x master mix 12.5 μl, upstream primer (20 pm) 1 μl, downstream primer (20 pm) 1 μl, probe (10 pm) 1 μl, template DNA 5 μl, and deionized water 7.5 μl.

[0024] Preferably, the multiplex qPCR reaction program is pre-denaturation 95℃ 5 min; 95℃ 30 s, 60℃ 45 s, 75℃ 45 s, 45 cycles; 75℃ 10 min, 4℃ 6 h.

[0025] The advantages of the present application are as follows: the present application distinguishes and identifies the main pathogenic microorganisms in ship ballast water, designs multiplex qPCR detection primers and probes according to the conserved regions, constructs a multiplex detection system, and can realize the detection and identification of various ship ballast water pathogenic microorganisms through one reaction. The detection system has good specificity and detection sensitivity, and the minimum can reach 2×10 -1 copies / μl, greatly shortens the detection time of the conventional PCR, realizes efficient and rapid identification, and has a great application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a multiplex qPCR sensitivity detection result graph. In the graph, 1 represents 2×10 5 copies / μL, 2 represents 2×10 4 copies / μL, 3 represents 2×10 3 copies / μL, 4 represents 2×10 2 copies / μL, 5 represents 2×10 1 copies / μL, 6 represents 2×10 0 copies / μL, and 7 represents 2×10 -1 copies / μL. DETAILED DESCRIPTION

[0027] The present application will be further described in detail below in combination with specific embodiments, so that those skilled in the art can more clearly understand the present application.

[0028] The following examples are intended to be illustrative of the present application and are not intended to limit the scope of the present application. Based on the specific examples in the present application, all other examples obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present application.

[0029] In the embodiments of the present application, all raw material components are commercially available products well known to those skilled in the art, unless otherwise specified. In the embodiments of the present application, the technical means used are conventional means well known to those skilled in the art, unless otherwise specified.

[0030] Example 1

[0031] 1.1 Primer design and synthesis

[0032] According to the icaa gene of Staphylococcus epidermidis, the ermx gene of Cutibacterium acnes, the stx2 gene of Escherichia coli, the vana gene of Enterococcus faecium, and the tlp gene of Vibrio alginolyticus included in ncbi as targets, multiple qPCR primers were designed using primer design software according to their conserved segments. The detection primers are as follows.

[0033] Staphylococcus epidermidis primer sequence:

[0034] 5'-ataggtggtttatggaaacaaagg-3' (seq id no. 1);

[0035] 5'-aacatagtacgatgtagacccatg-3' (seq id no. 2);

[0036] Probe sequence: 5'-fam-tgggctcaaggcgggcatgaagtact-bhq1-3' (seq id no. 11).

[0037] Cutibacterium acnes primer sequence:

[0038] 5'-tggttcacatttcacctgg-3' (seq id no. 3);

[0039] 5'-aaaggctttgcgctgctc-3' (seq id no. 4);

[0040] Probe sequence: 5'-hex-ccaaggtctgctttccggccacagccaaa-bhq1-3' (seq id no. 12).

[0041] E. coli primer sequences:

[0042] 5'-actgggggcgaatcagcaa-3' (seq id no. 5);

[0043] 5'-tcattcacagcgcgaacagaa-3' (seq id no. 6);

[0044] Probe sequence: 5'-tet-agtataggggagaggatggtgtcagagt-bhq1-3' (seq id no. 13).

[0045] S. faecalis primer sequences:

[0046] 5'-ccacctcgccaacagctaa-3' (seq id no. 7);

[0047] 5'-tgttaagccggcgcgttc-3' (seq id no. 8);

[0048] Probe sequence 5'-joe-ttcccaataccgcacaaccgacctcaca-bhq1-3' (seq id no. 14).

[0049] V. alginolyticus primer sequences:

[0050] 5'-ggatagaagttatcaagccctg-3' (seq id no. 9);

[0051] 5'-gtaatatggcagagtttccgtg-3' (seq id no. 10);

[0052] Probe sequence 5'-rox-tcagtacccattgctctgtcagcatcgttc-bhq1-3' (seq id no. 15).

[0053] 1.2 Preparation of pathogen standards

[0054] The upstream and downstream amplification primers of each pathogenic bacteria were used to amplify the icaa gene of Staphylococcus epidermidis, the ermx gene of Cutibacterium acnes, the stx2 gene of Escherichia coli, the vana gene of Enterococcus faecium, and the tlp gene of Vibrio alginolyticus by conventional PCR, and the PCR amplification products were recovered and purified by gel, then connected to the pgm-t vector, transfected into dh5α E. coli, and cultured. The positive recombinant bacteria were screened by blue-white spot and PCR, and the recombinant plasmid was extracted by plasmid extraction kit. The plasmid concentration was quantified by nanodrop2000, and the concentration was converted to copy number by Avogadro's constant. The recombinant plasmid with calculated copy number was diluted to a concentration of 2×10 7 The pathogenic bacteria standard was prepared, and stored at -70℃ for standby.

[0055] Example 2

[0056] 2.1 Pathogenic bacteria DNA extraction

[0057] The impurities in the ship ballast water were filtered conventionally, and the bacteria in the ballast water were quantitatively enriched by bacterial culture plate. After enrichment, the genomic DNA in the sample was extracted by bacterial genomic DNA extraction kit (dp302), and stored at -20℃ for standby.

[0058] 2.2 Multiple qPCR reaction establishment, see Table 1 for specific reaction system.

[0059] Table 1 Multiple qPCR reaction system

[0060]

[0061] 2.3 Multiple qPCR reaction program, see Table 2 for details.

[0062] Table 2 Multiple qPCR reaction program

[0063]

[0064] After the reaction, the fluorescence signal intensity in each channel was recorded by the enzyme marker, which was used to judge whether the ship ballast water contained the above pathogenic bacteria.

[0065] Example 3

[0066] 3.1 Specific detection

[0067] The detection system constructed in Example 2 was used to detect Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Streptococcus faecium, Vibrio alginolyticus, and other common pathogens such as Vibrio cholerae, Staphylococcus aureus, Actinomycetes, Pseudomonas, and cyanobacteria with the same nucleic acid concentration as the detection objects to analyze the specificity of the detection method.

[0068] The results are shown in Table 3 below. Except for Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Streptococcus faecium, and Vibrio alginolyticus, which showed obvious fluorescent signals, no other bacteria showed fluorescent signals, confirming that the multiplex qPCR detection method of the present invention is specific only to the target virus and has no cross-reaction with other detection objects, thus having good specificity.

[0069] Table 3. Specificity of detection

[0070]

[0071] 3.2 Sensitivity testing

[0072] The pathogen standard prepared in Example 1 was used as the object, and the pathogen standard was diluted 10 times with 1× PBS buffer to prepare 2×10 5 -2×10 -1 Each concentration gradient was set up with 3 replicates, and nuclease-free water was used as a template-free control. The detection limit of this method for the above pathogens was determined based on the multiplex qPCR reaction system to evaluate the sensitivity of the method. Figure 1 The results showed that the minimum detection limit of Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Streptococcus faecium, and Vibrio alginolyticus can reach 2×10 -1 copies / μl.

[0073] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the essence and scope of protection of the technical solutions of the present invention.

Claims

1. A kit for rapid detection of pathogenic microorganisms in ship ballast water, characterized in that: The kit contains a multiplex qPCR reagent for simultaneously detecting Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Streptococcus faecium, and Vibrio alginolyticus. The multiplex qPCR reagent includes specific amplification primers and probes. The sequences of the specific amplification primers are shown in SEQ ID NOs. 1-10, and the sequences of the specific amplification probes are shown in SEQ ID NOs. 11-15.

2. The kit according to claim 1, wherein The multiplex qPCR reagent further includes Multiplex PCR 5X Master Mix, deionized water and / or template DNA.

3. A multiplex qPCR reagent for rapid detection of pathogenic microorganisms in ship ballast water, characterized in that: The multiplex qPCR reagent includes specific amplification primers and probes for simultaneously detecting Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Streptococcus faecium, and Vibrio alginolyticus. The specific amplification primer sequences are shown in SEQ ID NOs. 1-10, and the specific amplification probe sequences are shown in SEQ ID NOs. 11-15.

4. The reagent according to claim 3, wherein The multiplex qPCR reagent further includes Multiplex PCR 5X Master Mix, deionized water and / or template DNA.

5. Use of the multiplex qPCR reagent according to any one of claims 3 to 4 in the preparation of a kit for detecting pathogenic microorganisms in ship ballast water.

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