Rapid detection method for vibrio corallinolyticus based on digital PCR (Polymerase Chain Reaction)

Abstract

The invention discloses a rapid detection method for vibrio corallinolyticus based on digital PCR, and relates to the technical field of biological detection.The method comprises the following steps that primers and probes are designed and synthesized according to the pyrH gene of the vibrio corallinolyticus; the method comprises the following steps: extracting total DNA of a sample to be detected as template DNA, and configuring a reaction system; and segmenting the prepared reaction system into micro-droplets by using a micro-droplet segmentation technology, then setting a reaction program to carry out PCR amplification, and calculating the content of the pyrH gene in the sample to be detected in combination with an end point fluorescence signal statistical result. According to the method, the pyrH gene is detected based on a digital PCR technology to reflect the pathogenic activity of pathogenic bacteria, the specificity and accuracy of vibrio corallinolyticus detection are remarkably improved, and an efficient, economical and accurate technical means is provided for comprehensive prevention and control of coral diseases.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a rapid detection method for Vibrio corallilyticus based on digital PCR. Background Art

[0002] Coral reefs are one of the most productive and biodiverse ecosystems in the ocean and are known as the "oasis of the ocean." They only account for 0.1% of the world's ocean area, but provide habitats for more than 25% of marine life and are breeding grounds and shelters for countless fish, crustaceans and invertebrates. However, in the past 30 years, about 50% of the world's coral reefs have been severely degraded due to climate change, ocean acidification, eutrophication and pathogen infection. Among them, coral diseases have exploded in tropical and subtropical waters, becoming the second largest cause of coral death after bleaching events. For example, the staghorn coral population in the Caribbean has degenerated by about 80% due to "white band disease" and "black band disease", while Australia's Great Barrier Reef has degenerated due to Vibrio corallolyticus ( Vibrio coralliilyticus Diseases caused by infections with coral reefs have spread to multiple regions. The accelerated loss of coral reefs not only threatens the balance of marine ecosystems but also threatens the collapse of coastal economies and the depletion of bioresources. Therefore, there is an urgent need to develop effective monitoring and control technologies for coral pathogens.

[0003] Coral diseases are caused by the combined effects of multiple pathogens, including bacteria, fungi, viruses, and protozoa. Outbreaks are often closely linked to environmental stresses, such as high temperatures and pollution. Among them, the Gram-negative bacterium Vibrio corallilyticus is currently one of the most destructive coral pathogens. This Gram-negative bacterium belongs to the Vibrio family and has an optimal growth temperature of 27°C-30°C. Its pathogenicity increases significantly with increasing seawater temperature. V. corallilyticus directly attacks coral tissue by secreting various virulence factors (such as metalloproteinases, hemolysins, and lipopolysaccharides), causing host cell lysis and the escape of symbiotic zooxanthellae (Symbiodiniaceae), ultimately leading to coral bleaching and tissue necrosis. Studies have shown that the pathogenicity of V. corallilyticus is temperature-dependent: it remains dormant below 25°C, but rapidly activates its virulence genes (such as toxR and vcpA) at temperatures reaching 30°C, causing coral mortality exceeding 90% within 48 hours.

[0004] In addition to Vibrio corallilyticus, other pathogens such as Vibrio shiloi (causing coral bleaching), Aurantimonas coralicida (causing white blight) and fungi Aspergillus sydowii(which induces sea fan disease) has also been linked to various coral diseases. However, Vibrio corallilyticus, due to its wide host range (infecting a wide range of coral hosts, including stony and soft corals), high environmental adaptability (tolerance to high salinity and high temperatures), and rapid spread, has become a core target for coral disease monitoring and prevention. Notably, existing detection technologies generally struggle to accurately identify low-abundance Vibrio corallilyticus in the early stages of disease, leading to delayed prevention and control measures and exacerbating irreversible damage to coral ecosystems.

[0005] Currently, the detection technologies for coral pathogens are mainly divided into three categories: traditional culture methods, molecular biology methods (PCR / qPCR) and metagenomic sequencing technology, but all of the above methods have significant defects. Traditional culture methods rely on selective culture media (such as TCBS agar) to isolate and culture coral-soluble Vibrio, which takes up to 3-7 days and is limited in sensitivity by multiple factors: first, only 1%-10% of bacteria in the marine environment can be detected by culture, and coral-soluble Vibrio can easily enter a "viable non-culturable state (VBNC)" under stress conditions, resulting in frequent false negative results; second, the culture medium may also enrich non-pathogenic Vibrio (such as V.alginolyticus ), requiring further differentiation through biochemical identification (such as the API 20E system), a cumbersome process and lacking specificity. While molecular detection techniques (PCR or qPCR) have shortened detection times to a few hours, they still face significant challenges. First, conventional PCR is susceptible to interference from inhibitors such as polysaccharides and humic acid in coral samples (e.g., mucus and tissue), resulting in reduced amplification efficiency or even failure, leading to high false-negative rates. Second, while qPCR allows for relative quantification, its reliance on standard curves and Ct values ​​makes it poorly suited for complex environmental samples. Variation in the genome copy number of coral pathogens (e.g., 16S rRNA copy number can vary up to 10-fold between strains) and the distorted effects of inhibitors on amplification curves can lead to quantitative errors of one to two orders of magnitude. While metagenomic sequencing can comprehensively profile the coral microbiome, it is costly and lacks sensitivity for detecting low-abundance pathogens (<0.1% of the community), making it unsuitable for rapid on-site screening.

[0006] Therefore, developing a more efficient and accurate method to detect Vibrio corallilyticus is of great significance for the early diagnosis of coral diseases and the protection of coral reef ecology. Summary of the Invention

[0007] In order to solve the above technical problems, the purpose of the present invention is to provide a rapid detection method for Vibrio corallilyticus based on digital PCR. This method uses digital PCR technology to detect the pyrH gene to reflect the pathogenic activity of the pathogen, significantly improving the specificity and accuracy of Vibrio corallilyticus detection, and providing an efficient, economical and precise technical means for the comprehensive prevention and control of coral diseases.

[0008] The present invention solves the above technical problems with the following technical solutions: providing a rapid detection method for Vibrio corallilyticus based on digital PCR, comprising the following steps: (1) Design and synthesize primers and probes based on the pyrH gene of Vibrio corallilyticus; (2) Extract the total DNA of the sample to be tested as template DNA and prepare the reaction system; (3) The configured reaction system is divided into droplets using droplet segmentation technology, and then the reaction program is set to perform PCR amplification. The pyrH gene content in the sample to be tested is calculated based on the statistical results of the end-point fluorescence signal.

[0009] Furthermore, in step (1), the nucleotide sequence of the pyrH gene is shown as SEQ ID NO.1.

[0010] Furthermore, in step (1), the primers include an upstream primer and a downstream primer, whose nucleotide sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0011] Furthermore, in step (1), the nucleotide sequence of the probe is shown as SEQ ID NO.4.

[0012] Furthermore, the 5' end of the probe is connected to a fluorescent group, and the 3' end is connected to a quencher group that matches the fluorescent group.

[0013] Furthermore, the fluorescent group and the quenching group are FAM and BHQ1, respectively.

[0014] Furthermore, in step (2), the reaction system includes template DNA, upstream primers, downstream primers, probes, ddPCR premix and double-distilled water.

[0015] Furthermore, the ddPCR premix includes DNA polymerase, buffer, dNTPs and Mg 2+ .

[0016] Furthermore, the 20 μL reaction system contained 100 ng of template DNA, 1.8 μL of upstream primer, 1.8 μL of downstream primer, 0.5 μL of probe, and 10 μL of 2× ddPCR premix, with the remainder being double-distilled water.

[0017] Furthermore, the concentrations of the upper primer, downstream primer, and probe were 10 μM.

[0018] Furthermore, in step (3), the reaction procedure was as follows: initial denaturation at 95°C for 10 min; denaturation at 94°C for 30 s, annealing / extension at 58.8°C for 1 min, and 40 cycles.

[0019] Furthermore, detection was performed without template addition as a negative control, and detection was performed using a plasmid containing the pyrH gene as a template DNA as a positive control.

[0020] The present invention also provides the use of the above primers and probes in preparing a digital PCR detection product for Vibrio corallilyticus.

[0021] The present invention also provides a kit for digital PCR detection of Vibrio corallilyticus, comprising the above primers and probes.

[0022] The present invention has the following beneficial effects: 1. This invention utilizes digital PCR (dPCR) technology, which partitions the reaction system into 20,000-100,000 droplets for independent amplification. Combined with endpoint fluorescence signal statistics, this method achieves absolute quantification without the need for a standard curve, demonstrating significant advantages across the board for coral pathogen detection. First, digital PCR achieves single-copy sensitivity, with a 10-100-fold improvement in the detection limit for low-abundance Vibrio corallilyticus compared to qPCR. Second, the droplet segmentation technique employed by digital PCR dilutes inhibitor concentrations in the sample, significantly reducing false negatives. Third, digital PCR can directly count positive droplets, avoiding the influence of amplification efficiency bias in qPCR.

[0023] 2. The pyrH gene is highly conserved and associated with virulence. The present invention uses the pyrH gene as the target gene, analyzes the conserved regions of the gene, and designs specific primers and probes based on the conserved regions, which can effectively distinguish closely related bacterial species, significantly improve the detection specificity, and increase the positive detection rate and accuracy.

[0024] 3. The detection method of this invention shortens the sample pre-processing time to 1 hour, and the total detection cycle takes only 4 hours, which is much shorter than traditional culture methods (3-7 days) and metagenomic sequencing (several days of analysis). In addition, the cost of a single test is low (only 10% of metagenomic sequencing), which is suitable for on-site rapid screening needs and provides efficient, economical and accurate technical support for the comprehensive prevention and control of coral diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 1 is the result of digital PCR detection using different primers; Figure 2 It is the temperature gradient scatter plot of digital PCR; Figure 3 This is a digital PCR scatter plot of sample detection. DETAILED DESCRIPTION

[0026] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0027] Example 1 A rapid detection method for Vibrio corallilyticus based on digital PCR, comprising the following steps: (1) Primers and a probe were designed and synthesized based on the pyrH gene (SEQ ID NO.1) of Vibrio corallilyticus; the upstream primer and the downstream primer were shown in SEQ ID NO.2 and SEQ ID NO.3, respectively; the probe included a nucleotide fragment shown in SEQ ID NO.4, a FAM fluorescent group was connected to the 5' end, and a BHQ1 quenching group was connected to the 3' end; (2) Extract the total DNA of the sample to be tested as template DNA and prepare the reaction system; the reaction system includes template DNA, upstream primers, downstream primers, probes, 2× ddPCR premix and double-distilled water; (3) The prepared reaction system was divided into droplets using droplet segmentation technology. The reaction program was then set (initial denaturation at 95°C for 10 min; denaturation at 94°C for 30 s, annealing / extension at 58.8°C for 1 min, 40 cycles) for PCR amplification. The pyrH gene content in the sample to be tested was calculated based on the statistical results of the endpoint fluorescence signal.

[0028] Example 2 A kit for digital PCR detection of Vibrio corallilyticus, comprising primers and probes, wherein the upstream primer and the downstream primer are shown as SEQ ID NO. 2 and SEQ ID NO. 3, respectively; the probe comprises a nucleotide fragment shown as SEQ ID NO. 4, and has a FAM fluorescent group connected to the 5' end and a BHQ1 quenching group connected to the 3' end.

[0029] Experimental Example 1 Primer Efficiency Test Referring to the detection method of Example 1, the pyrH gene amplification primers reported by previous researchers (upstream primer: 5'-CAACTGGGCAGACGCAATCCGTGAGT-3', downstream primer: 5'-CGTAAATACGCCATCAACTTTTGTC-3') and the primers designed by this application (SEQ ID NO. 2 and SEQ ID NO. 3) were used respectively. Both primer sets used the probe sequence designed by this application. The DNA extracted from the diseased coral tissue pieces in the reef area was used as the template for detection. The results are as follows: Figure 1 As shown, the left figure is the primer test results reported by previous researchers, and the right figure is the primer test results in Example 1.

[0030] Depend on Figure 1 It can be seen that when the same DNA template was used for detection, the primers reported by previous researchers detected 19 positive clones, while the primers in Example 1 detected 38 positive clones. It can be seen that the detection rate of the primers and probes in Example 1 is higher.

[0031] Test Example 2 Optimal Annealing Temperature Test The pyrH gene was amplified using diseased coral DNA as a template and ligated to a T vector to obtain a DNA plasmid containing the target fragment, which was used as a positive standard in turn.

[0032] Referring to the detection method of Example 1, the annealing temperatures were set at 55°C, 55.7°C, 56.9°C, 58.8°C, 61.1°C, 63°C, 64.3°C and 65°C, a total of 8 temperature gradients, to detect the positive standard. The results are as follows: Figure 2 shown.

[0033] Depend on Figure 2 As can be seen, positive and negative scatter points are further apart at 55°C-61.1°C. Combined with the positive copy number reference, the copy number is highest at 58.8°C. To ensure probe specificity, 58.8°C was selected as the optimal annealing temperature for droplet digital PCR.

[0034] Test Example 3 Sample Testing Referring to the detection method of Example 1, six field-collected reef-building coral DNA samples (including diseased corals and healthy corals, S1-S6) were tested, with no template added and positive standards as negative (-) and positive (+) controls. The test results are as follows: Figure 3 The results showed that this method can effectively detect reef-building coral samples, determine positive results based on positive droplets, and determine the pathogenic activity of the pathogen Vibrio corallilyticus.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A rapid detection method for Vibrio corallilyticus based on digital PCR, characterized in that: The following steps are involved: (1) Design and synthesize primers and probes based on the pyrH gene of Vibrio corallilyticus; (2) Extract the total DNA of the sample to be tested as template DNA and prepare the reaction system; (3) Using droplet segmentation technology, the configured reaction system is divided into droplets, and then a reaction program is set to perform PCR amplification, and the pyrH gene content in the sample to be tested is calculated based on the statistical results of the end-point fluorescence signal.

2. The rapid detection method for Vibrio corallilyticus based on digital PCR according to claim 1, characterized in that: In step (1), the nucleotide sequence of the pyrH gene is shown in SEQ ID NO.

1.

3. The rapid detection method for Vibrio corallilyticus based on digital PCR according to claim 1, characterized in that: In step (1), the primers include an upstream primer and a downstream primer, and their nucleotide sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

4. The rapid detection method for Vibrio corallilyticus based on digital PCR according to claim 1, characterized in that: In step (1), the nucleotide sequence of the probe is shown in SEQ ID NO.

4.

5. The rapid detection method for Vibrio corallilyticus based on digital PCR according to claim 1, characterized in that: In step (2), the reaction system includes the template DNA, upstream primers, downstream primers, probes, ddPCR premix and double-distilled water.

6. The rapid detection method for Vibrio corallilyticus based on digital PCR according to claim 1, characterized in that: In step (3), the reaction procedure is: initial denaturation at 95°C for 10 min; denaturation at 94°C for 30 s, annealing / extension at 58.8°C for 1 min, and 40 cycles.

7. The rapid detection method for Vibrio corallilyticus based on digital PCR according to claim 1, characterized in that: At the same time, detection without template addition was used as a negative control, and detection using a plasmid containing the pyrH gene as a template DNA was used as a positive control.

8. Use of the primers and probes described in claim 1 in preparing a digital PCR detection product for Vibrio corallilyticus.

9. A kit for digital PCR detection of Vibrio corallilyticus, characterized in that: The method comprises the primers and probes according to claim 1.

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