Burkholderia gladioli coconut poison pathogenic variant test strip detection method, composition and kit

Abstract

The invention relates to a body temperature amplification test strip rapid detection method of Burkholderia gladioli coconut poison pathogenic variants in food. The invention also relates to an oligonucleotide primer probe composition for the method. The invention also relates to a detection kit containing the composition. When the composition is used for test strip body temperature amplification detection, burkholderia gladioli coconut poison pathogenic variant components in a food sample can be simply, rapidly, specifically and sensitively detected.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection, and in particular, relates to a rapid detection technology of a temperature amplification test strip for gladiolus Burkholderia cocos parasites, an oligonucleotide primer probe composition used in the method, and a kit containing the composition. Background Art

[0002] Burkholderia gladiolus ( Burkholderia gladioli ) belongs to the Burkholderia family, Burkholderia genus, and is an aerobic or facultative anaerobic non-fermenting Gram-negative bacillus widely found in the environment. Among them, Burkholderia gladiolus pv. cocovenenum ( Burkholderia gladioli Pathovar cocovenenans ) can secrete a variety of toxins including fumonisin. Ingestion of food contaminated with Burkholderia gladioli pv. cocovenenum can lead to severe food poisoning, with the main symptoms being nausea, vomiting, diarrhea, convulsions, unconsciousness, and multiple organ failure such as liver and kidney, and eventually death from respiratory failure. It is the foodborne pathogen with the highest mortality rate discovered so far. Most cases of food poisoning caused by Burkholderia gladioli pv. cocovenenum are related to fermented rice flour, wet starch and other foods.

[0003] In recent years, poisoning incidents caused by accidentally eating food contaminated with Burkholderia gladioli pv. cocovenenans have occurred frequently.

[0004] With the rapid development of molecular biology technology, various molecular biology techniques that trace biological components by identifying nucleic acids have the advantages of strong specificity, high sensitivity, not easily disturbed by environmental conditions, and stable results. They are increasingly widely used in the detection of foodborne pathogenic microorganisms. However, the currently popular PCR technology usually takes about 2 hours to detect, and requires a professional variable temperature nucleic acid amplifier. Enzymatic isothermal amplification (ERA) is a new isothermal amplification technology developed in 2019 and is an alternative to polymerase chain reaction (PCR). It has the same specificity as PCR amplification, but is faster. It can usually amplify the nucleic acid template to a detectable level in 7 to 10 minutes; and the ERA reaction does not require thermal denaturation, so it does not require expensive thermal cycling equipment. In addition, ERA requires a lower temperature than other isothermal amplification techniques, and can complete the exponential amplification of trace DNA / RNA segments at 37 to 42°C, so it is also called body temperature amplification technology.

[0005] Under normal temperature, the recombinase and primer bind tightly to form a polymer of recombinase and primer. When the recombinase and primer polymer search for a complementary sequence that matches the template DNA, the double-stranded structure of the template DNA is opened with the help of single-stranded DNA binding protein. Then, under the action of DNA polymerase, the primer extends along the 5'→3' direction to form a new complementary DNA chain and complete the exponential growth of the amplified product. It has made major breakthroughs in low-temperature adaptability and sensitivity and has reached the international advanced level. In addition, this technology is an isothermal nucleic acid amplification technology developed by my country with global independent intellectual property rights, which can get rid of foreign patent barriers. This technology is convenient, accurate and rapid, and has become a hot topic of research in recent years. Summary of the invention

[0006] The purpose of the present invention is to quickly, efficiently, simply, intuitively, accurately and sensitively detect gladiolus Burkholderia cocos venom pathogenic variants in food samples. The method is of great significance for the on-site rapid screening and risk detection of gladiolus Burkholderia cocos venom pathogenic variants, and can effectively prevent food poisoning incidents caused by accidental ingestion of gladiolus Burkholderia cocos venom pathogenic variants.

[0007] The inventors of the present invention identified the Burkholderia gladioli pv. B. gladioli Toxigenic genes of Co14 Bon M, and retrieved the gene sequences of non-pathogenic gladiolus Burkholderia and other common foodborne pathogens, such as Salmonella, Klebsiella, and Yersinia enterocolitica. According to the design principle of ERA test strip primers and probes, specific test strip primers and probes that can efficiently detect gladiolus Burkholderia cocos venom pathogenic variants were designed and screened in the inter-genus difference region. A rapid test strip detection technology for gladiolus Burkholderia cocos venom pathogenic variants was established.

[0008] In one aspect of the present invention, a test strip is provided for detecting gladiolus Burkholderia cocos parasites, the upstream oligonucleotide primer F2-2: 5'-GCGATGGTCCGTATCTCCTGCTTGTGCG-3' (SEQ ID No. 1), and the downstream R2-1: 5'-GACAGGTTCCAGTGCCATTACGTGCCGC-3' (SEQ ID No. 2), and the 5' end of the downstream primer is modified with Biotin. In another aspect of the present invention, a test strip is also provided for detecting gladiolus Burkholderia cocos parasites, the probe P2: 5'-TGTCCTGGATCTCCACCAGCTCGAAACCGGCATCCTTCAGCAGCTTCG-3' (SEQ ID No. 3), the 5' end of the probe is modified with a FAM group, the 3' end is blocked with a c3-spacer, and the 33rd T base is replaced with THF. The probe was combined with SEQ ID No. 1 and SEQ ID No. 2 for test strip amplification, thereby specifically identifying gladiolus Burkholderia cocos parasites, and the reaction was carried out at a constant temperature of 37° C. for 20 min.

[0009] In another aspect of the present invention, a composition comprising the above oligonucleotide sequence is provided, which comprises the oligonucleotide primer pairs SEQ ID No. 1 to SEQ ID No. 2 for the Burkholderia gladioli pv. cocovenenans test strip detection and the probe SEQ ID No. 3 sequence.

[0010] In one embodiment, the amplification condition of the test strip for gladiolus Burkholderia cocos parasites is a constant temperature reaction at 37 ° C for 20 min. After the reaction is completed, 5 μL of the reaction product is taken to a 1.5 mL centrifuge tube and diluted 25 times with pure water; take out the test strip (do not touch the NC membrane), insert it into the centrifuge tube, wait for the test strip to be completely soaked by the liquid, and read the result according to the color development of the control band and the test band after 3 to 10 minutes. If both the control band and the test band have obvious red lines, it means that the sample is positive and contains the target bacterial nucleic acid component; if only the control band has a red line, it means that the sample is negative, does not contain the target bacterial nucleic acid component or the content is below the detection limit; if there is no red line on the control band, it is determined that the test result is invalid.

[0011] In one aspect of the present invention, a kit for detecting Burkholderia cerevisiae pv. cocovenenans by an ERA test strip method is provided, wherein the kit comprises the oligonucleotide sequence or the composition.

[0012] The kit provided by the invention comprises a specific primer-probe combination for rapid detection of gladiolus Burkholderia cocos parasites by an ERA test strip method, and a user manual.

[0013] In a specific embodiment, the kit for detecting gladiolus Burkholderia of the present invention further comprises a reference substance. Preferably, the reference substance comprises a positive control substance and a negative control substance. In one embodiment, the negative control substance is sterile double distilled water.

[0014] In one embodiment, the sensitivity of the ERA test strip method for detecting Burkholderia gladioli pv. -2 ng / μL.

[0015] In yet another aspect, the present invention provides use of the composition or the kit in detecting Burkholderia gladioli pv. cocovevenenan in food samples.

[0016] The design of primer probes is the key to developing sensitive and rapid ERA test strip detection methods, because primer probes with different sequences have different performances in the test strip reaction, which will affect the amplification efficiency and amplification speed, so primer design and screening are very necessary.

[0017] Since ERA amplification technology has a unique reaction condition of 37-42 ℃, ERA detection has strict requirements for primer design. Ordinary PCR primers are not applicable because ERA primers are longer than general PCR primers, usually 28-35 nt. Primers that are too short will reduce the recombination rate, affect the amplification speed and detection sensitivity. If the primers are too long, other secondary structures such as primer dimers or hairpin structures may be generated during the amplification process, thereby affecting the yield of nucleic acid amplification. And when designing ERA primers, the denaturation temperature is no longer a key factor affecting the amplification primers.

[0018] In order to establish a rapid detection method for ERA test strips, it is necessary to further design the detection probe. The probe is 46-52 nt long, of which at least 30 nt is located at the 5' end of the THF site, and at least 15 nt is located at its 3' end. THF replaces a base located 30 bp downstream of the fluorophore and 15 bp upstream of the blocking group. The fluorescent group can only be labeled on thymine (T). The 3' end of the probe needs to be modified and blocked with a blocking group, such as C3-spacer, phosphate, amine, biotin or tetraethylene glycol.

[0019] The design of primers and probes for ERA is not as mature as that of traditional PCR, and there is currently no design software available. Although the requirements for the design of primers and probes are known, the final selection still needs to be screened and optimized by yourself, and the experimental results are not predictable.

[0020] According to the characteristics of toxin production of Burkholderia cocosus pathogenic strains of gladiolus, the inventors have determined that the toxin-producing gene BonM was used as a specific gene, and primers and probes for ERA test strips were designed based on multiple specific sites on the gene, forming four combinations: F1 / R1-1 / P1, F1 / R1-2 / P1, F2-2 / R2-1 / P2, and F2-2 / R2-2 / P2. The modification groups and sequence information are detailed in Table 1. The primer and probe combinations were compared and analyzed based on the test strip test results. By comparing the amplification efficiency ( Figure 1 ), specificity ( Figure 2 ), detection limit ( Figure 3 ), as well as negative control and other factors, the oligonucleotide primer probe combination F2-2 / R2-1 / P2 for detecting gladiolus Burkholderia cocos parasites by the ERA test strip method provided by the present invention was finally determined.

[0021] Table 1 Sequence information of primers and probes designed for detection of Burkholderia gladiolus ERA

[0022] The method of the present invention cleverly utilizes the specificity of ERA technology for efficient DNA amplification and nucleic acid hybridization, and combines the rapidity, sensitivity and naked eye visibility of the test strip to establish an on-site visual rapid detection method that is simple to operate, time-saving and labor-saving, reliable in results, and accurate and sensitive. The ERA test strip detection technology of the present invention can be used for the qualitative detection of gladiolus Burkholderia in food samples in a simple, rapid, specific and sensitive manner. The establishment of this patented method can provide good technical support for on-site rapid risk screening of gladiolus Burkholderia. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the analysis result of the amplification efficiency of the designed 4 sets of ERA test strips. In the figure, AD represents the amplification results of the primer probe combination F1 / R1-1 / P1, F1 / R1-2 / P1, F2-2 / R2-1 / P2, and F2-2 / R2-2 / P2 on five species of gladiolus Burkholderia, 1 represents gladiolus Burkholderia CGMCC1.2892, 2 represents gladiolus Burkholderia CGMCC1.3360, 3 represents gladiolus Burkholderia pv. cocosus CICC25108, 4 represents gladiolus Burkholderia pv. cocosus CICC25126, 5 represents gladiolus Burkholderia pv. cocosus CICC25132, CK represents ddH2O blank control, and the experiment was repeated 3 times.

[0024] Figure 2The results are as follows: specific analysis results of the ERA test strip method for Burkholderia gladioli by the preferred primer-probe combination F2-2 / R2-1 / P2; the control samples are Burkholderia gladioli CICC25108, Escherichia coli O157:H7IQCC10193, Escherichia coli IQCC10138, Shigella sonnei ATCC29930, Yersinia enterocolitica ATCC27729, Listeria monocytogenes IQCC22291, Staphylococcus aureus CMCC26003, Bacillus cereus ATCC10876, Bacillus subtilis ATCC11774, Vibrio vulnificus ATCC27562, and beta-hemolytic Streptococcus CMCC32210; CK represents the ddH2O blank control, and the experiment was repeated 3 times.

[0025] Figure 3 The results are the analysis results of the sensitivity of the preferred primer-probe combination F2-2 / R2-1 / P2 to the ERA test strip method for Burkholderia gladiolus. The template concentrations were 10 2 , 10 1 , 10 0 , 10 -1 , 10 -2 ng / μL, and the experiment was repeated 3 times.

[0026] Table 2 shows the test results of the preferred primer-probe combination F2-2 / R2-1 / P2 on 15 purchased food samples, and the comparison with the test results of GB 4789.29-2020. Sterile ddH2O was used as a blank control, 2 parallel replicates were performed for each sample, and the experiment was repeated 3 times. DETAILED DESCRIPTION

[0027] The present invention is further described by way of examples, but the present invention is not limited to the following examples.

[0028] Example 1 Screening of primer-probe combinations of ERA test strips for detecting Burkholderia gladioli pv. cocosvenorensis.

[0029] Four sets of probes were designed (Table 1). Through the analysis of amplification efficiency, sensitivity and specificity, the ERA test strip primer probes for the detection of Burkholderia cerebrospinalis pv. cocosinus were finally determined.

[0030] 1) DNA extraction of Burkholderia gladiolus: using PrepMan TM DNA of Burkholderia gladiolus was extracted using Ultra Sample preparation Reagent kit and diluted to 10 ng / μL with sterile water for testing.

[0031] 2) Detection system: Prepare the premix for each sample according to the instructions of the test paper type nucleic acid amplification kit (ERA method): 20 μL of solvent, 2.1 μL of forward primer, 2.1 μL of reverse primer, 0.6 μL of probe, 1 μL of template, 2.2 μL of ddH2O2. Transfer the above premix to the PCR tube with the test paper type amplification reagent, oscillate to mix and centrifuge briefly. Add 2 μL of activator to the tube cap, carefully cover the tube cap, centrifuge briefly to allow the activator to enter the premix, oscillate briefly to mix and centrifuge again quickly, and keep the temperature at 37 °C for 20 min. At the same time, use sterile water as a blank control.

[0032] 3) Result determination: If obvious red lines appear in both the control band and the test band, it means that the sample is positive and contains target bacterial nucleic acid components; if only the control band has a red line, it means that the sample is negative and does not contain target bacterial nucleic acid components or the content is below the detection limit; if no red line appears in the control band, the test result is judged to be invalid.

[0033] like Figure 1 As shown, the amplification efficiency of four combinations of candidate primers and probes for the ERA test strip detection of Burkholderia cerebrospinalis pv. cocovenenans was compared. The results showed that the amplification efficiency of the primer-probe combination F2-2 / R2-1 / P2 was the best.

[0034] like Figure 2 As shown, the specificity analysis of the screened primer probe combination F2-2 / R2-1 / P2 showed that only gladiolus Burkholderia caerulea pv. cocosvenorensis was amplified, while other gladiolus Burkholderia caerulea and common foodborne pathogenic bacteria DNA and ddH2O blank control were not amplified, which fully demonstrated that the specific oligonucleotide primers screened in this experiment showed excellent specificity for the detection of gladiolus Burkholderia caerulea pv. cocosvenorensis.

[0035] like Figure 3 As shown in the figure, the sensitivity of the screened primer probe combination F2-2 / R2-1 / P2 was further analyzed. The results showed that the oligonucleotide primer combination had the highest sensitivity, and the minimum content of Burkholderia cerebrospinalis pv. cocovesum that could be detected was 10 -2 ng / μL.

[0036] Example 2 Analysis of the accuracy and applicability of the selected Burkholderia gladioli pv. cocosvenorensis ERA test strip primer probes for rapid detection of actual commercially available samples.

[0037] 1) Sample pretreatment: Take 25 g of commercially available sample and add it to a conical flask containing 225 mL of GVC enrichment solution. After shaking culture at 37 °C and 130 rpm for 24 h, perform DNA extraction test.

[0038] 2) DNA extraction, detection system, reaction procedure and result determination steps are the same as those in Example 1.

[0039] The actual commercial results are shown in Table 2.

[0040] Table 2 Actual test results of commercially available samples

[0041]

[0042] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the claims and their equivalents.

Claims

1. A composition for detecting gladiolus Burkholderia cocos parasites by ERA test strip method, It is characterized in that The composition includes a primer pair 5'-GCGATGGTCCGTATCTCCTGCTTGTGCG-3' and 5'-GACAGGTTCCAGTGCCATTACGTGCCGC-3' for detecting gladiolus Burkholderia cocos parasites, and a probe 5'-TGTCCTGGATCTCCACCAGCTCGAAACCGGCATCCTTCAGCAGCTTCG-3', wherein the 5' end of the probe is modified with a FAM group, the 33rd T base is replaced with THF, and the 3' end is blocked with a c3-spacer.

2. A method for rapidly detecting Burkholderia cerebrospinalis pv. cocosvenorensis by ERA test strip method, the method comprising using the composition of claim 1.

3. A kit for rapid detection of gladiolus Burkholderia cocos parasites by ERA test strip method, the kit comprising the composition according to claim 1.

4. Use of the composition of claim 1, the method of claim 2, and the kit of claim 3 in the detection of Burkholderia cerebrovelin pv. cocosvenorensis on gladiolus.

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