Primer combination for multiple detection kit of marine pathogenic bacteria and multiple detection kit

Abstract

The invention provides a primer combination for a multiple detection kit for marine pathogenic bacteria and the multiple detection kit. The primer combination comprises primer pairs and probes which are used for detecting 13 marine pathogenic bacteria: vibrio alginolyticus, vibrio parahaemolyticus, vibrio cholerae, vibrio vulnificus, vibrio marine fish, vibrio river, vibrio menivibrio, vibrio mimicus, vibrio harveyi, vibrio occultus, vibrio horii, plesiomonas and aeromonas, and the sequence of the primer combination is as shown in SEQ1-SEQ39. According to the present invention, the genomic DNA of 13 marine pathogenic bacteria is identified and detected in one digital PCR reaction through the ultra-multiple digital PCR fluorescence coding technology, and the kit not only covers the common vibrio species such as vibrio cholerae, vibrio vulnificus, vibrio parahaemolyticus and the like, but also can identify the sea area vibrio, vibrio holii, vibrio occultus and the like, and the kit has good sensitivity, specificity and accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and particularly to a primer combination and a multiplex detection kit for multiplex detection of marine pathogenic bacteria. Background Art

[0002] Marine pathogenic bacteria mainly include Vibrio parahaemolyticus, Vibrio anguillarum, Vibrio alginolyticus, Vibrio harveyi, etc., which refer to microorganisms existing in the marine environment and capable of causing diseases in humans, animals or plants. They are widely distributed in seawater, sediments, the surfaces and bodies of marine organisms, and may be transmitted through direct contact, consumption of contaminated seafood, or wound exposure, etc. For example, some marine pathogenic bacteria are important pathogens of zoonotic diseases. Humans can be infected with pathogenic bacteria by eating contaminated seafood or contacting seawater with damaged skin, which can cause diseases such as otitis media, urinary tract infection, septicemia, etc., and even death.

[0003] Therefore, rapid, comprehensive and accurate detection of marine pathogenic bacteria is an important way to protect human health and reduce economic losses. Therefore, there is a need for multiplex detection of marine pathogenic bacteria in this field. Summary of the Invention

[0004] The object of the present invention is to provide a primer combination and a multiplex detection kit for multiplex detection of marine pathogenic bacteria.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides a primer combination for a multiplex detection kit for marine pathogenic bacteria, and the primer combination includes primer pairs and probes for detecting 13 kinds of marine pathogenic bacteria; the 13 kinds of marine pathogenic bacteria include: Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio damsela, Vibrio fluvialis, Vibrio metschnikovii, Vibrio mimicus, Vibrio harveyi, Vibrio cincinnatiensis, Vibrio hollisae, Plesiomonas and Aeromonas; the sequences of the primer pairs and probes in the primer combination are shown as SEQ1-SEQ39.

[0007] Preferably, the probe is labeled with a fluorescent group or a quenching group at the 5'-end and / or 3'-end.

[0008] Preferably, the fluorescent group is any one or more of FAM, HEX, ROX, CY5; the quenching group is any one or more of BHQ1, BHQ2, BHQ3.

[0009] Preferably, in order to achieve multiplex detection, the probes for detecting the same marine pathogenic bacteria include probes labeled with different fluorescent groups.

[0010] Preferably, the fluorescence labeling of the probes in the primer combination is shown in Table 1.

[0011] The present invention provides a kit for multiplex detection of marine pathogenic bacteria, and the kit includes the primer combination as described above.

[0012] Preferably, the kit further includes a detection Buffer, a detection enzyme, a positive control product, a negative control product, and an MgCl2 solution.

[0013] Preferably, the detection Buffer is a dPCR Buffer; the detection enzyme is a dPCR Taq enzyme; the positive control product is a mixed solution of an artificially synthesized gene containing the target sequence of the marine pathogenic bacteria; the negative control product is nuclease-free water.

[0014] The present invention provides a detection method for a kit for multiplex detection of marine pathogenic bacteria, and the detection method includes the following steps:

[0015] (1) Extract the DNA of the sample to be tested;

[0016] (2) Perform digital PCR reaction using the primer combination according to any one of claims 1-3;

[0017] (3) Combine the multi-color fluorescence coding and decoding technology to perform typing detection on the results.

[0018] Preferably, when performing digital PCR reaction, the detection channels are selected as FAM, HEX, ROX, Cy5, and Quasar705; the Qusara705 is a human endogenous reference channel; the sequences of the primer pair and probe of the internal reference are shown in SEQ40-SEQ42.

[0019] Preferably, in the digital PCR reaction system, the final concentration of each probe in the primer combination is 100-400 nM, and the final concentration of each primer is 400-600 nM.

[0020] Preferably, the sample to be tested is a marine environmental sample, a marine organism-related sample (such as feces, blood, etc. of marine organisms), or a wound secretion, feces, blood, etc. sample from a clinical patient (such as a patient after being injured during offshore operations).

[0021] The beneficial effects of the technical solution of the present invention:

[0022] The present invention provides a primer combination and a multiplex detection kit for a marine pathogenic bacteria multiplex detection kit. Through the ultra-multiplex digital PCR fluorescence encoding technology, genomic DNA of 13 kinds of marine pathogenic bacteria, namely Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Photobacterium damselae, Vibrio fluvialis, Vibrio metschnikovii, Vibrio mimicus, Vibrio harveyi, Vibrio cincinnatiensis, Grimontia hollisae, Plesiomonas, and Aeromonas, are identified and detected in one digital PCR reaction. The kit of the present invention not only covers common Vibrio species such as Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus, but also can identify uncommon Vibrio species such as Photobacterium damselae, Grimontia hollisae, and Vibrio cincinnatiensis. At the same time, the primer combination and the kit of the present invention have good sensitivity, specificity, and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is an example of the four-color fluorescence encoding detection results of FAM, HEX, ROX, and CY5 of the present invention;

[0024] In the figure, all positive points in the four channels are located at the same position, that is, it is determined that the template to be tested is the template corresponding to the 4-color encoded target;

[0025] Figure 2 It is an example of the results of the FAM single-color encoded target and the HEX+ROX double-color encoded target of the present invention;

[0026] In the figure, the positive points in the HEX and ROX channels are located at the same position, and it can be confirmed as the detection result of the double-color encoded target. The positive points in the FAM channel are in different positions from the positive points in the HEX and ROX channels. Therefore, it is confirmed as the detection result of the FAM channel single-color encoded target.

[0027] Figure 3 It is the test result of the negative sample of the primer combination of the present invention.

[0028] Figure 4 It is the target test result of the primer combination of the present invention.

[0029] Figure 5Results of primer-probe optimization study designed for Aeromonas

[0030] Figure 6 Results of primer-probe optimization study designed for Vibrio vulnificus

[0031] Figure 7 Results of primer-probe optimization study designed for Plesiomonas Detailed implementation manners

[0032] The following further elaborates on the detailed implementation manners of the present invention in conjunction with embodiments. Unless otherwise specified, the instruments and equipment involved in the embodiments are all conventional instruments and equipment; the raw materials involved are all commercially available conventional raw materials; the test methods involved are all conventional methods.

[0033] Example 1 Design of primer-probe system

[0034] Design the primer combination (primer pairs and probes) according to the conserved regions of marine pathogenic bacteria (as shown in Table 1). The specific method is as follows: ① Download the whole genome dataset of the species from NCBI; ② Screen for species-specific genes (Blast+); ③ Analyze the conservation of specific genes (Blast+); ④ Select the most conserved region for primer and probe design (analyzed by IDT Oligo Analyzer). As can be seen from Table 2, the coverage rate of the primer-probe pairs designed by the present invention for 13 marine pathogenic bacteria all reached over 95%; ⑤ Analyze the specificity and dimer of multiple primers and probes according to MFE Primer.

[0035] Table 1 Sequence information corresponding to primer combinations

[0036]

[0037]

[0038]

[0039]

[0040]

[0041] Table 2 Target coverage rate of primer-probe combinations

[0042] species coverage number coverage rate Vibrio parahaemolyticus 420 / 423 99.29％ Vibrio cholerae 782 / 794 98.49％ Vibrio alginolyticus 417 / 420 99.29％ Vibrio vulnificus 533 / 535 99.63％ Vibrio fluvialis 114 / 114 100.00％ Plesiomonas 38 / 38 100.00％ Aeromonas 145 / 146 99.32％ Vibrio metschnikovii 84 / 84 100.00％ Vibrio mimicus 29 / 29 100.00％ Vibrio harveyi 67 / 70 95.71％ Vibrio damsela 67 / 68 98.53％ Vibrio cincinnatiensis 19 / 19 100.00％ Vibrio hollisae 10 / 10 100.00％

[0043] Example 2 A kit for multiplex detection of marine pathogenic bacteria

[0044] The multiplex detection kit for marine pathogenic bacteria of the present invention combines digital PCR multiplex coding technology, can break through the channel limitation, and achieve multiplex detection of foodborne pathogenic bacteria. Among them, genomic DNA of a total of 13 marine pathogenic bacteria, namely Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio damsela, Vibrio fluvialis, Vibrio metschnikovii, Vibrio mimicus, Vibrio harveyi, Vibrio cincinnatiensis, Vibrio hollisae, Plesiomonas and Aeromonas, can be identified and detected. The multiplex detection kit for marine pathogenic bacteria of the present invention is applicable to the BioDigital series digital PCR platform, and when in use, it needs to be combined with a digital PCR oil phase and consumables matching the BioDigital digital PCR system. The digital PCR reaction system and reaction program are shown in Table 2 and Table 3.

[0045] The present invention provides a multiplex detection kit for marine pathogenic bacteria, comprising: the primer combination described in Example 1, a buffer for detecting marine pathogenic bacteria (3×Maxuseful dPCR Buffer), an enzyme for detecting marine pathogenic bacteria (dPCR Taq enzyme), a positive control product (a mixed solution of linearized plasmids containing target fragments of marine pathogenic bacteria synthesized artificially), a negative control product (nuclease-free water), a digital PCR chip, a digital PCR oil phase, and an MgCl2 solution (100 mM).

[0046] Table 3 Digital PCR reaction system

[0047]

[0048]

[0049] Note: The final concentration of each primer in the primer combination of the reaction system is 400 - 600 nM; the final concentration of each probe in the primer combination of the reaction system is 100 nM.

[0050] Table 4 Digital PCR reaction program

[0051]

[0052] Example 3 A detection method for multiplex detection of marine pathogenic bacteria

[0053] The present invention provides a detection method for a kit for multiplex detection of marine pathogenic bacteria, comprising the following steps:

[0054] (1) Extract the DNA of the sample to be tested:

[0055] Use a nucleic acid purification and extraction kit to extract and purify the genomic DNA from the wound sample of a patient after being injured during offshore operations as the DNA of the sample to be tested.

[0056] Among them, the sample concentration is adjusted according to the sample type and controlled at 105 copies / reaction or less.

[0057] (2) Prepare the reaction system:

[0058] Using the kit for multiplex detection of marine pathogenic bacteria described in Example 2, configure the digital PCR reaction system containing primer combinations respectively; mix well and centrifuge to enrich the liquid at the bottom of the tube; perform digital PCR reaction according to the reaction procedure;

[0059] (3) Combine the multi-color fluorescence encoding and decoding technology to perform genotyping detection on the results;

[0060] Among them, when performing digital PCR reaction, the detection channels are selected as FAM, HEX, ROX, Cy5, and Quasar705. Among them, Qusara705 is the human internal reference channel, and it may be detected as negative for environmental samples;

[0061] (4) Data collection and analysis

[0062] Threshold setting: First, adjust the threshold line in the one-dimensional scatter plot. The threshold line can be automatically performed by the software or manually adjusted: just exceeding the highest point of the negative droplets.

[0063] Data analysis: The digital PCR reaction system uses multi-color coding for result interpretation. When there is more than one positive result channel, it is necessary to confirm whether it is a multiple infection through the two-dimensional scatter plot results.

[0064] (5) Positive judgment value

[0065] ① The positive judgment values of each detection target of this kit are shown in Table 5 below:

[0066] Table 5 Positive judgment values of each detection target of this kit

[0067] serial number number of positive droplets in FAM / HEX / ROX / Cy5 channels result determination 1 ≥3 positive 2 ＜3 negative

[0068] ② According to the positive and negative results of each channel, refer to Table 6 (Note: Only single pathogen infection situations are shown in the table) for pathogen detection result interpretation:

[0069] Table 6

[0070] FAM HEX ROX CY5 Quasar705 identification result + - - - - Vibrio alginolyticus - + - - - Vibrio parahaemolyticus - - - + - Vibrio cholerae - - + - - Plesiomonas + + - - - Aeromonas + - - + - Vibrio fluvialis + - + - - Vibrio metschnikovii - + - + - Vibrio mimicus - - + + - Vibrio harveyi - + + - - Vibrio vulnificus + - + + - Vibrio damsela + + - + - Vibrio cincinnatiensis - + + + - Vibrio hollisae - - - - + internal reference

[0071] Experimental Example 1 Test results of negative samples of the primer combination of the present invention

[0072] Using the primer combination designed in Example 1, perform dPCR detection on the nucleic acid samples extracted from negative fecal samples or negative human tissue samples according to the detection method of Example 3. The results are as Figure 3As shown, it can be seen that the specific results all meet the requirements. Experimental Example 2 Target test results of the primer combination of the present invention

[0073] The characteristic genes of marine pathogenic bacteria corresponding to the primer sets designed in Example 1 were cloned and constructed into plasmids, and were respectively diluted in a 10-fold gradient within the range of 10 - 10000 copies / ul for linear testing. According to the detection method of Example 3, the templates of each marine pathogenic bacterium diluted in gradient were respectively detected, and the positive one-dimensional scatter plot of digital PCR is as Figure 4 shown, and the results show that all templates have good detection effects.

[0074] Experimental Example 3 Optimization research on the primers and probes of the present invention

[0075] During the design process of the primers and probes in Example 1 of the present invention, a series of different primer and probe combinations were tested to screen for primer and probe combinations that can achieve better technical effects. As shown in Table 7, the primer and probe combination (highlighted) designed in Example 1 of the present invention has better technical effects compared to other available primer and probe combinations.

[0076] Table 7 Comparison of the effects of different primer and probe combinations

[0077]

[0078]

[0079]

[0080]

[0081] For example, two sets of primers and probes designed for Aeromonas were respectively tested using plasmids, as Figure 5 shown. The positive signals of the two sets of sequences are not very different, but the amplification efficiency of group 2 is more stable, and combination 2 is comprehensively and preferably selected.

[0082] Two sets of primers and probes designed for Vibrio vulnificus were respectively tested using plasmids, as Figure 6 shown. The signal-to-noise ratio of group 1 is too low, and the positive and negative droplets can hardly be distinguished. Combination 2 is comprehensively and preferably selected.

[0083] Two sets of primers and probes designed for Plesiomonas were respectively tested using plasmids, as Figure 7 shown. The positive amplification of group 1 is abnormal, and combination 2 is comprehensively and preferably selected.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, for those of ordinary skill in the art, it is still possible to modify the technical solutions recorded in the foregoing embodiments, or to equivalently replace some of the technical features thereof; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. A primer combination for multiplex detection of marine pathogenic bacteria, characterized in that, The primer combination includes primer pairs and probes for detecting 13 species of marine pathogenic bacteria; the 13 species of marine pathogenic bacteria include: Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio damsela, Vibrio fluvialis, Vibrio metschnikovii, Vibrio mimicus, Vibrio harveyi, Vibrio cincinnatiensis, Vibrio hollisae, Plesiomonas and Aeromonas; the sequences of the primer pairs and probes in the primer combination are shown as SEQ1-SEQ39.

2. The primer combination according to claim 1, wherein The probe is labeled with a fluorescent group or a quenching group at the 5'-end and / or 3'-end.

3. The primer combination according to claim 2, wherein The fluorescent group is any one or more of FAM, HEX, ROX, CY5; the quenching group is any one or more of BHQ1, BHQ2, BHQ3.

4. A kit for multiplex detection of marine pathogenic bacteria, characterized in that, The kit includes the primer combination according to any one of claims 1-3.

5. The kit according to claim 4, characterized in that, The kit further includes a detection Buffer, a detection enzyme, a positive control product, a negative control product, and an MgCl2 solution.

6. The kit according to claim 5, wherein The detection Buffer is a dPCR Buffer; the detection enzyme is a dPCR Taq enzyme; the positive control product is a mixed solution of an artificial synthetic gene containing the marine pathogenic bacteria target sequence according to claim 1; the negative control product is nuclease-free water.

7. A detection method for multiplex detection of marine pathogenic bacteria, characterized in that, The detection method includes the following steps: (1) Extract the DNA of the sample to be tested. (2) Perform digital PCR reaction using the primer combination according to any one of claims 1-3. (3) Combine the multi-color fluorescence decoding technology to perform typing detection on the results.

8. The detection method according to claim 7, wherein When performing digital PCR reaction, the detection channels are selected as FAM, HEX, ROX, Cy5 and Quasar705; the Qusara705 is a human endogenous reference channel; the sequences of the primer pair and probe of the reference are shown as SEQ40-SEQ42.

9. The detection method according to claim 7, characterized in that In the digital PCR reaction system, the final concentration of each probe in the primer combination is 100-400 nM, and the final concentration of each primer is 400-600 nM.

10. The detection method according to claim 7, wherein The sample to be tested is a marine environmental sample, a marine organism-related sample, or a wound secretion, feces or blood sample from a clinical patient.

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