Detection method for pathogenic microorganisms in ship ballast water and application thereof

Abstract

The invention discloses a method for detecting pathogenic microorganisms in ship ballast water and application of the method, and belongs to the technical field of biology. According to the present invention, the main pathogenic microorganisms in the ship ballast water are distinguished and identified, the multiple qPCR detection primer probes are designed according to the conserved region, the multiple detection system is constructed, the detection and the identification of the multiple pathogenic microorganisms in the ship ballast water can be achieved through the one-time reaction, and the detection system has characteristics of good specificity and good detection sensitivity, the method has the advantages that the detection time of conventional PCR (polymerase chain reaction) is greatly shortened, high-efficiency and rapid identification is realized, and the method has a relatively great application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for detecting pathogenic microorganisms in ship ballast water and its application. Background Art

[0002] Ship ballast water refers to the water and suspended substances added to a ship to control the ship's trim, list, 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 process of following the ship's voyage because they cannot adapt to changes in factors such as temperature and salinity, but some can survive and eventually be discharged into a new environment along with the ship ballast water. As a result, a biological community or a variety of organisms in one water area is transmitted to another geographically isolated water area with the ballast water. If these organisms can grow, reproduce and establish populations in natural or semi-natural ecosystems or habitats due to the lack of natural enemies or other reasons, they may threaten the ecological system structure and species diversity of these bays, estuaries or inland waters, becoming invasive alien species. Moreover, ballast water can also spread harmful parasites and pathogens, and may even lead to the extinction of local species.

[0003] With the continuous growth of the international shipping industry and the continuous enhancement of people's awareness of marine environmental protection, the problem of alien marine biological invasion caused by the discharge of ship ballast water has attracted extensive attention from all sectors of society. Recognizing that the cross-regional transfer of harmful aquatic organisms and pathogens in ship ballast water has had a non-negligible impact on the global marine ecological environment, the Global Environment Facility (GEF) has listed it as one of the four major threats to the ocean. In summary, it is particularly important to establish the detection and identification of pathogenic microorganisms in ship ballast water. Summary of the Invention

[0004] Aiming at the technical problems existing in the prior art, the present invention provides a method for detecting pathogenic microorganisms in ship ballast water and its application. The present invention distinguishes and identifies the main pathogenic microorganisms in ship ballast water, designs multiplex qPCR detection primers and probes according to their conserved regions, constructs a multiplex detection system, and can realize the detection and identification of various 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 prospects.

[0005] The primary object of the present invention is to provide a kit for rapidly detecting pathogenic microorganisms in ship ballast water, and the kit contains multiplex qPCR reagents for Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Streptococcus faecium and Vibrio alginolyticus simultaneously.

[0006] Preferably, the multiplex qPCR reagents include specific amplification primers and probes.

[0007] Preferably, the specific amplification primer sequences are successively as shown in SEQ ID NO. 1-10.

[0008] Preferably, the specific amplification probe sequences are successively as shown in SEQ ID NO. 11-15.

[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 invention is to provide a multiplex qPCR reagent for rapid detection of pathogenic microorganisms in ship ballast water, wherein the multiplex qPCR reagent comprises specific amplification primers and probes for Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Streptococcus faecium and Vibrio alginolyticus simultaneously.

[0011] Preferably, the specific amplification primer sequences are successively as shown in SEQ ID NO. 1-10.

[0012] Preferably, the specific amplification probe sequences are successively as shown in SEQ ID NO. 11-15.

[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 invention is to provide the use of the multiplex qPCR reagent in the preparation of a kit for detecting pathogenic microorganisms in ship ballast water, wherein the multiplex qPCR reagent comprises specific amplification primers and probes for simultaneously detecting Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Streptococcus faecium and Vibrio alginolyticus.

[0015] Preferably, the specific amplification primer sequences are successively as shown in SEQ ID NO. 1-10.

[0016] Preferably, the specific amplification probe sequences are successively as shown in SEQ ID NO. 11-15.

[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 invention is to further 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 the following detection method, and the method comprises the following steps:

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

[0021] 2) using the DNA as a template, performing a multiplex qPCR reaction using the multiplex qPCR reagent or the kit;

[0022] 3) Use an enzyme-labeled instrument to record the fluorescence signal in the product.

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

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

[0025] The advantages of the present invention are as follows: the present invention distinguishes and identifies the main pathogenic microorganisms in ship ballast water, and designs multiple qPCR detection primers and probes based on their conserved regions to construct a multiple detection system, which can realize the detection and identification of multiple ship ballast water pathogenic microorganisms in a single reaction. The detection system has good specificity and detection sensitivity, which can reach a minimum of 2×10 -1 copies / μl, which greatly shortens the detection time of conventional PCR, realizes efficient and rapid identification, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the result of multiplex qPCR sensitivity test. In the figure, 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, 7 represents 2×10 -1 copies / μL. DETAILED DESCRIPTION

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

[0028] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

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

[0030] Example 1

[0031] 1.1 Primer design and synthesis

[0032] Multiplex qPCR primers were designed using primer design software based on conserved regions of the NCBI-listed Staphylococcus epidermidis icaa gene, Propionibacterium acnes ermx gene, Escherichia coli stx2 gene, Streptococcus faecium vana gene, and Vibrio alginolyticus tlp gene. The detection primers are shown below.

[0033] Primer sequences for Staphylococcus epidermidis:

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

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

[0036] Probe sequence: 5'-FAM-TGGGCTCAAGGCGGCATGAAGTACT-BHQ1-3' (Seq ID no. 11).

[0037] Primer sequences for Propionibacterium acnes:

[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] Primer sequences for Escherichia coli:

[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] Primer sequences for Streptococcus faecium:

[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] Primer sequences for Vibrio alginolyticus:

[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] Using the upstream and downstream amplification primers of each pathogen, 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 were subjected to conventional PCR amplification. After the PCR amplification products were recovered and purified by gel, they were ligated into the pGM-T vector, transfected into DH5α Escherichia coli for culture, and positive recombinant bacteria were screened by blue-white screening and PCR. The recombinant plasmid was extracted using a plasmid extraction kit. The concentration of the plasmid was quantified using a nucleic acid analyzer Nanodrop2000, and the concentration was converted to the copy number using Avogadro's constant. The recombinant plasmid with the calculated copy number was diluted to a concentration of 2×10 7 copies / μl to prepare a pathogen standard product, which was stored at -70°C for later use.

[0055] Example 2

[0056] 2.1 Pathogen DNA extraction

[0057] The impurities in the ship's ballast water were filtered conventionally, and the bacteria in the ballast water were quantitatively enriched using a bacterial culture plate. After enrichment, the genomic DNA in the sample was extracted using a bacterial genomic DNA extraction kit (DP302) and stored at -20°C for later use.

[0058] 2.2 Establishment of multiplex qPCR reaction, and the specific reaction system is shown in Table 1.

[0059] Table 1 Multiplex qPCR reaction system

[0060]

[0061] 2.3 Multiplex qPCR reaction program, which is specifically shown in Table 2.

[0062] Table 2 Multiplex qPCR reaction program

[0063]

[0064] After the reaction, a microplate reader was used to record the fluorescence signal intensity in each channel to determine whether the above-mentioned pathogens were present in the ship's ballast water.

[0065] Example 3

[0066] 3.1 Specificity 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 method for detecting pathogenic microorganisms in ship ballast water, characterized in that, The method includes a multiplex qPCR reagent for simultaneously detecting Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Streptococcus faecium, and Vibrio alginolyticus.

2. The method according to claim 1, characterized in that The multiplex qPCR reagent includes specific amplification primers and specific amplification probes.

3. The method according to claim 2, wherein The sequences of the specific amplification primers are shown as SEQ ID NO.1-10 in sequence.

4. The method according to claim 2, wherein The sequences of the specific amplification probes are shown as SEQ ID NO.11-15 in sequence.

5. The method according to claim 2, characterized in that, The multiplex qPCR reagent further includes multiplex PCR 5xmaster mix, deionized water, and / or template DNA.

6. 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.

7. The reagent according to claim 6, wherein The sequences of the specific amplification primers are shown as SEQ ID NO.1-10 in sequence.

8. The reagent according to claim 6, wherein, The sequences of the specific amplification probes are shown as SEQ ID NO.11-15 in sequence.

9. The reagent according to claim 6, wherein The multiplex qPCR reagent further includes multiplex PCR 5xmaster mix, deionized water, and / or template DNA.

10. Use of the multiplex qPCR reagent according to any one of claims 6-9 in the preparation of a kit for detecting pathogenic microorganisms in ship ballast water.

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