CN118389720B - A primer-probe combination, kit and method for detecting foodborne Clostridium - Google Patents

Abstract

The invention discloses a primer probe combination, a kit and a method for detecting food-borne clostridium, and belongs to the technical field of pathogenic clostridium detection. The invention is based on a real-time fluorescence quantitative PCR technology, and the primer probe composition which has good amplification specificity and high sensitivity and does not generate amplification interference in an amplification system is obtained by screening and optimizing the primer and the probe, so that the detection and quantitative analysis of clostridium botulinum, clostridium difficile, clostridium perfringens and clostridium butyricum are simultaneously realized.

Description

A primer-probe combination, kit and method for detecting foodborne Clostridium

Technical Field

The present invention belongs to the technical field of pathogenic clostridium detection, and in particular relates to a primer-probe combination, a kit and a method for detecting foodborne clostridium.

Background Art

Foodborne diseases are one of the major challenges facing global public health. These diseases are usually caused by ingesting food or water contaminated by pathogens. Among them, Clostridium is a group of important foodborne pathogens, including Clostridium botulinum , Clostridium difficile , Clostridium perfringens and Clostridium butyricum .

Although there are many methods for detecting Clostridium, such as culture, immunological and molecular biological methods, these methods often have shortcomings such as long detection time, complex operation, limited sensitivity and specificity. The culture method is the traditional gold standard, but it requires a long time to culture, and its sensitivity and specificity are insufficient for some Clostridium species with demanding conditions. Immunological methods, such as enzyme-linked immunosorbent assay (ELISA), are faster than culture methods, but may be affected by cross-reactions, reducing specificity. Molecular biological methods, especially polymerase chain reaction (PCR) technology, have improved detection sensitivity and specificity. Conventional PCR can achieve rapid detection of microorganisms, especially pathogens, but cannot achieve quantitative detection and has low detection efficiency; quantitative PCR can quantitatively detect pathogens to be detected, but only one pathogen to be detected can be detected in one experiment, and high-throughput detection of pathogens cannot be achieved. Real-time fluorescent quantitative PCR (qPCR) technology refers to the method of adding fluorescent groups to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally using the standard curve to quantitatively analyze unknown templates. qPCR can quantitatively detect genetic nucleic acids of pathogenic pathogens in real time, and therefore has advantages over immunological methods such as chemiluminescence, time resolution, and protein chips.

Chinese patent document CN101928773A discloses an oligonucleotide primer, detection method and use for detecting common pathogens based on fluorescent quantitative PCR technology, wherein the pathogens are pathogens present in blood, sputum, water, food, atmosphere and soil environment. For example, Chinese patent documents CN109022550A, CN109022603A, CN110055311A and CN111518923A disclose a method for detecting type A clostridium botulinum, type B clostridium botulinum, clostridium difficile and clostridium perfringens based on fluorescent quantitative PCR technology respectively. However, there is no report in the prior art on the simultaneous detection and quantification of clostridium botulinum, clostridium difficile, clostridium perfringens and clostridium butyricum using fluorescent quantitative PCR technology. Based on this, the present invention provides a method for simultaneously detecting and quantifying Clostridium botulinum, Clostridium difficile, Clostridium perfringens and Clostridium butyricum based on fluorescent quantitative PCR technology. The present invention significantly improves the amplification efficiency, has good sensitivity and specificity, and realizes accurate detection and quantitative analysis by screening and optimizing primer probes.

Summary of the invention

One object of the present invention is to provide a primer-probe combination for detecting foodborne Clostridium; a second object of the present invention is to provide the use of the primer-probe combination in the preparation of a product for detecting foodborne Clostridium; a third object of the present invention is to provide a kit for detecting foodborne Clostridium; and a further object of the present invention is to provide a method for detecting foodborne Clostridium.

If not otherwise specified, the foodborne Clostridium described in the present invention is a combination of Clostridium botulinum , Clostridium difficile , Clostridium perfringens and Clostridium butyricum .

The purpose of the present invention is achieved through the following technical solutions:

In a first aspect, the present invention provides a primer-probe combination for detecting foodborne Clostridium, wherein the primer-probe combination is composed of upstream and downstream primers and probes shown in 1)-4):

1) The upstream and downstream primers for detecting Clostridium botulinum are shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the probe is shown in SEQ ID NO: 3;

2) The upstream and downstream primers for detecting Clostridium difficile are shown in SEQ ID NO: 4 and SEQ ID NO: 5, and the probe is shown in SEQ ID NO: 6;

3) The upstream and downstream primers for detecting Clostridium perfringens are shown in SEQ ID NO: 7 and SEQ ID NO: 8, and the probe is shown in SEQ ID NO: 9;

4) The upstream and downstream primers for detecting Clostridium butyricum are shown as SEQ ID NO: 10 and SEQ ID NO: 11, and the probe is shown as SEQ ID NO: 12.

Furthermore, the 5' end of the probe is labeled with a fluorescent reporter group, and the fluorescent reporter signals of the probes do not interfere with each other; the 3' end of the probe is labeled with a fluorescent quencher group.

The "signals do not interfere with each other" described in the present invention means that the fluorescent groups used in the above-mentioned probes are different and will not affect each other's detection, that is, different channels can be used for detection. For example, FAM, HEX, ROX, and Cy5 can be used. The absorbance values of these groups are not close, and different channels can be selected. The signals of different channels will not interfere with each other; in addition, the three signals of each fluorescent channel can be distinguished by adding different probe concentrations to distinguish the fluorescence values, thereby forming different clustering signals, and further distinguished by cluster analysis software, without mutual interference.

In a specific embodiment of the present invention, the fluorescent reporter groups labeled at the 5' end of the probes used to detect the four foodborne Clostridium species are selected from FAM, HEX, ROX and Cy5, respectively.

In a second aspect, the present invention provides a use of the primer-probe combination provided in the first aspect of the present invention in preparing a product for detecting foodborne Clostridium.

The products for detecting foodborne Clostridium include but are not limited to reagents, test kits, chips, test papers, membrane strips or detection platforms.

In a third aspect, the present invention provides a kit for detecting foodborne Clostridium, the kit comprising the primer-probe combination described in the first aspect of the present invention.

Furthermore, the kit also includes an enzyme mixture, a negative quality control product, a positive quality control product, and a PCR buffer.

The enzyme mixture contains at least one of high-fidelity Taq enzyme, UDG enzyme, magnesium ions and dNTP.

The negative quality control product is pure water treated with ethylene glycol pyrocarbonate (DEPC); the positive quality control product is a plasmid containing the specific sequences of Clostridium botulinum , Clostridium difficile , Clostridium perfringens and Clostridium butyricum described in the present invention.

In a fourth aspect, the present invention provides a method for detecting foodborne Clostridium, the method comprising the following steps:

(1) Extract DNA or RNA from the sample to be tested;

(2) configuring a PCR amplification system containing the primer-probe combination described in the first aspect of the present invention to carry out a PCR amplification reaction;

(3) Determine whether the corresponding foodborne Clostridium exists in the sample to be tested and the initial concentration of the foodborne Clostridium based on the amplification curve.

In the amplification system described in step (2), the final concentration of the upstream primer is 0.1-0.2µM, the final concentration of the downstream primer is 0.2-0.3µM, and the final concentration of the probe is 0.1-0.15µM.

The sample to be tested described in the present invention is any substance that may contain foodborne Clostridium, including but not limited to food, feces, oral swabs, and anal swabs.

The detection method provided by the present invention is based on real-time fluorescence quantitative PCR technology. By detecting the strength of the fluorescence signal, the amplification amount of the PCR product can be detected in real time. Clostridium botulinum , Clostridium difficile , Clostridium perfringens and Clostridium butyricum can be detected and quantitatively analyzed simultaneously, and the detection sensitivity is high. Further, the present invention screens and optimizes the designed primers, selects primer pairs with good specificity and high sensitivity as candidates, eliminates primer groups that form primer dimers in the multiple PCR system, and finally preferably obtains a primer pair combination with strong specificity and good amplification effect to prepare a kit for detecting the four foodborne clostridia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the amplification curve of the stool sample of Example 4 on a fluorescent PCR instrument;

Figure 2 Amplification curve of the specific detection experiment with only Clostridium botulinum gene template added;

Figure 3 Amplification curve of the specific detection experiment with only Clostridium difficile gene template added;

FIG4 is an amplification curve diagram of the specific detection experiment in which only the Clostridium perfringens gene template is added;

Fig. 5 is the amplification curve of the specific detection experiment with only Clostridium butyricum gene template added;

Fig. 6 Amplification curve of the specific detection experiment with only Clostridium tetani gene template added;

Figure 7: Results of the Clostridium botulinum sensitivity test;

Figure 8: Results of the Clostridium difficile sensitivity test;

Figure 9 is a graph showing the results of a sensitivity test of Clostridium perfringens;

Figure 10 Result of sensitivity test of Clostridium butyricum;

FIG. 11 is a graph showing amplification curves of mutual interference in Example 7.

DETAILED DESCRIPTION

The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1 Primer-probe combination

The nucleotide information of the primer probe provided by the present invention is shown in the following table.

Table 1 Primer-probe combinations for detecting four foodborne Clostridium species

.

Unless otherwise specified, "F" in Table 1 represents an upstream primer; "R" represents a downstream primer; and "P" represents a probe.

Furthermore, the 5' end of the probe in the above table is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group. Specifically,

The probes for detecting Clostridium botulinum are:

5’-FAM-ATTAAGAAGCTATGATATGAAGGGAAAGAG-BHQ1-3’;

The probes for detecting Clostridium difficile are:

5’-HEX-GATTCTTCTGGAAATGTATAAGTTTC-BHQ1-3’;

Probes for detecting Clostridium perfringens:

5’-ROX-AGAAGGGAGCCTGACTGCGACACC-BHQ2-3’;

Probes for detecting Clostridium butyricum:

5’-Cy5-CATTATATAATTTACAATCTGGAGCAACGC-BHQ2-3’;

Example 2 Kit for detecting foodborne Clostridium

The present invention provides a kit as shown in the following table:

Table 2 Test kits for detecting foodborne Clostridium difficile

.

In the above kit, the final concentration of the upstream primer in the primer-probe mixture is selected from 0.1-0.2 µM, the final concentration of the downstream primer is selected from 0.2-0.3 µM, and the final concentration of the probe is selected from 0.1-0.15 µM. Those skilled in the art can prepare the enzyme mixture by conventional techniques or purchase it through commercial channels.

Example 3 Method for detecting foodborne Clostridium

(1) Obtaining samples for testing

(2) Extraction of DNA from samples to be tested

The genomic DNA in the sample to be tested is extracted using a commercially available DNA extraction kit or a method well known to those skilled in the art.

(3) PCR amplification reaction

a) mixing the primer-probe mixture and the enzyme mixture in the kit provided in Example 2 at a volume ratio of 1:5 to form a PCR amplification system, and dispensing into reaction tubes, wherein the final concentration of the upstream primer is 0.1 µM, the final concentration of the downstream primer is 0.2 µM, and the final concentration of the probe is 0.1 µM;

b) Add the nucleic acid extract of the sample to be tested (or negative or positive quality control) to the reaction tube, cover the tube tightly, centrifuge it momentarily, and put it into the thermal amplification instrument. Perform PCR amplification reaction according to the amplification program shown in the table below, and read the fluorescence in the corresponding channels of FAM, HEX, ROX, and Cy5 respectively.

Table 3 PCR amplification program

.

(4) Result determination

If there is no S-shaped curve in the amplification, the sample is judged to be negative;

If the amplification curve shows an obvious S-shape, it is judged as a positive sample.

The positive and negative quality control products provided in the kit are used for quality control in clinical trials, and the operation method is the same as that of the samples to be tested. The quality control standards are as follows:

The amplification curve of the negative control product has no obvious S-shaped growth; the amplification curve of the positive control product is S-shaped;

The above requirements must be met simultaneously in the same experiment, otherwise, the experiment will be considered invalid and must be repeated.

Example 4 Detection of fecal samples

(1) Sample collection, transportation and storage

Place the stool sample to be tested in a special dry small box. The storage period should not exceed one month. If necessary, transport it at room temperature.

(2) Sample processing

a) Take an appropriate amount of sample and place it in a centrifuge tube containing 300 μl Trizol and 100 μl chloroform. Fold the tube cap, shake vigorously for 20 seconds, let it stand for 3 minutes, and then centrifuge at 12,000 rpm for 2 minutes;

b) Carefully remove the colorless upper layer of liquid (be careful not to touch the middle flocculent layer), transfer it to a newly sterilized 1.5 ml centrifuge tube, then add 10 μl of magnetic beads (mix thoroughly and then aspirate), mix thoroughly with an oscillator, centrifuge at 8,000 rpm for 1 minute, and carefully discard all the liquid;

c) Add 400 μl of 75% DEPC ethanol, shake thoroughly to mix, centrifuge at 8,000 rpm for 1 minute, and drain as much liquid as possible (use the pipette tip to drain the residual liquid to avoid touching the precipitated particles);

d) Place the centrifuge tube containing the precipitate in a fume hood to air-dry for 15 minutes (you can also use an open heater to dry at 60°C for 5 minutes to prevent cross-contamination of samples), then add 30μl of nuclease-free _H2O , fully resuspend with an appropriate pipette, let stand for 1 minute, then centrifuge at 8,000rpm for 1 minute. The processed samples can be used directly for PCR detection or temporarily stored at -20°C. If stored for more than one month, they can be stored at -70°C.

The PCR amplification reaction was carried out using the method provided in Example 3. The test results are shown in FIG1 . As can be seen from the figure, all four foodborne Clostridium species in the stool samples tested in this example were positive.

Example 5 Kit-Specific Detection

In the quadruple PCR amplification system, only the Clostridium botulinum gene template was added, and the other three Clostridium templates were not added. PCR amplification was performed according to the method provided in Example 3. The results are shown in Figure 2. It can be seen that only the FAM channel showed an S-shaped amplification curve in the amplification results, and no amplification occurred in other channels.

In the quadruple PCR amplification system, only the Clostridium difficile gene template was added, and the other three Clostridium templates were not added. PCR amplification was performed according to the method provided in Example 3. The results are shown in Figure 3. It can be seen that only the HEX channel showed an S-shaped amplification curve in the amplification results, and no amplification occurred in other channels.

In the quadruple PCR amplification system, only the Clostridium perfringens gene template was added, and the other three Clostridium templates were not added. PCR amplification was performed according to the method provided in Example 3. The results are shown in FIG4 . It can be seen that only the ROX channel showed an S-shaped amplification curve in the amplification results, and no amplification occurred in other channels.

In the quadruple PCR amplification system, only the Clostridium butyricum gene template was added, and the other three Clostridium templates were not added. PCR amplification was performed according to the method provided in Example 3. The results are shown in Figure 5. It can be seen that only the Cy5 channel showed an S-shaped amplification curve in the amplification results, and no amplification occurred in other channels.

The above results indicate that the kit and detection method provided by the present invention can well distinguish the four pathogenic Clostridium species described in the present invention.

Clostridium tetani, which is relatively close to the four clostridia described in the present invention, was selected as the bacteria to be tested, and the Clostridium tetani gene template was added to the amplification system. PCR amplification was performed according to the method provided in Example 3. The results are shown in Figure 6. There was no obvious S-shaped growth in the amplification curve, indicating that the primer probe combination provided by the present invention had no cross-reaction with Clostridium tetani, which was similar in species to the four foodborne clostridia described in the present invention, and had good specificity.

Example 6 Kit Sensitivity Detection

Take a certain amount of positive plasmids containing the four Clostridium species described in the present invention and dissolve them in an appropriate amount of ultrapure water. By measuring the OD value, the concentration is calculated by the formula OD ₂₆₀ × 50µg/ml. The number of copies per milliliter is calculated according to the molar mass of the plasmid, and then the ultrapure water is used to dilute to 5×10 ³ copies/ml, 1×10 ³ copies/ml, 2×10 ² copies/ml, and 5×10 ¹ copies/ml. Since each plasmid contains 1 copy of the gene fragment of each Vibrio species, the concentration of each Vibrio species is the same.

In the reaction tube, add 10µl of enzyme mixture and 0.5µl of primer probe mixture. After preliminary mixing, add 10µl of plasmid template of various gradients respectively. Each gradient is repeated three times. The temperature control program of 50℃ for 2 minutes, 95℃ for 3 minutes, followed by 40 cycles (95℃ for 10 seconds, 58℃ for 35 seconds) is adopted. The experiment is carried out on the Hongshi SLAN-96S five-channel fluorescence quantitative PCR instrument.

The results of the sensitivity test of Clostridium botulinum are shown in Figure 7, FAM channel, three replicates, 5×10 ¹ copies/ml is negative; the results of the sensitivity test of Clostridium difficile are shown in Figure 8, HEX channel, three replicates, 5×10 ¹ copies/ml is negative; the results of the sensitivity test of Capsella perfringens are shown in Figure 9, ROX channel, three replicates, 5×10 ¹ copies/ml is negative; the results of the sensitivity test of Clostridium butyricum are shown in Figure 10, Cy5 channel, three replicates, 5×10 ¹ copies/ml is negative. Based on the above experimental results, the sensitivity of the foodborne Clostridium detection kit provided by the present invention is 2×10 ² copies/ml.

Example 7 Primer design and optimization process for detecting foodborne Clostridium

According to the prior art, each foodborne Clostridium gene or DNA fragment is obtained as a candidate amplification gene; multiple pairs of spare primers are designed for all candidate amplification genes or DNA fragments using premier6 software; all primer schemes are subjected to Blast analysis in Genbank to obtain primer schemes with higher specificity as experimental verification schemes (multiple candidate amplification genes will be selected for each Clostridium, and multiple pairs of spare primers will be designed for each candidate amplification gene. After Blast analysis, 1-2 experimental verification schemes appear for one candidate amplification gene of each Clostridium); single-plex PCR verification is performed on each experimental verification scheme to determine the availability of the primers, including specificity evaluation and sensitivity evaluation, and spare primers with poor effects are eliminated; all verified primers are mixed for multiplex PCR to verify the availability of the primers during co-amplification, and primer pairs that form primer dimers are removed, and finally the primer-probe combination for detecting four foodborne Clostridium provided in Example 1 of the present invention is obtained.

Since the four Clostridium genes described in the present invention are very similar, it is not easy to select specific regions, which often leads to poor specificity in the design of multiple primers. When the primers were first designed, it was found that when the Clostridium perfringens template was added, the amplification of Clostridium butyricum would also be brought about. After multiple optimizations, the specificity and mutual interference were eliminated. Exemplarily, the present invention provides a set of primer-probe combinations that have the above problems, as shown in the following table:

Table 4 Primer-probe combinations with mutual interference

.

In the quadruple PCR amplification system, only the Clostridium perfringens gene template was added, and the other three Vibrio templates were not added. PCR amplification was performed according to the method provided in Example 3. The results are shown in Figure 11. Due to poor primer design, mutual interference occurred in the amplification, and obvious amplification of Clostridium butyricum was observed in the amplification system with only Clostridium perfringens.

Finally, it should be noted that 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims (7)

1. A primer-probe combination for detecting foodborne Clostridium, the primer-probe combination consisting of upstream and downstream primers and probes shown in 1)-4): 1) The upstream and downstream primers for detecting Clostridium botulinum are shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the probe is shown in SEQ ID NO: 3; 2) The upstream and downstream primers for detecting Clostridium difficile are shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe is shown in SEQ ID NO:6; 3) The upstream and downstream primers for detecting Clostridium perfringens are shown in SEQ ID NO:7 and SEQ ID NO:8, and the probe is shown in SEQ ID NO:9; 4) The upstream and downstream primers for detecting Clostridium butyricum are shown in SEQ ID NO: 10 and SEQ ID NO: 11, and the probe is shown in SEQ ID NO: 12; The foodborne clostridium is a combination of Clostridium botulinum , Clostridium difficile , Clostridium perfringens and Clostridium butyricum . 2. The primer-probe combination according to claim 1, characterized in that the 5' end of the probe is labeled with a fluorescent reporter group, and the fluorescent reporter signals of the probes do not interfere with each other; the 3' end of the probe is labeled with a fluorescent quencher group. 3. The primer-probe combination according to claim 1, characterized in that the fluorescent reporter groups labeled at the 5' end of the probes for detecting Clostridium botulinum, Clostridium difficile, Clostridium perfringens and Clostridium butyricum are selected from FAM, HEX, ROX and Cy5, respectively. 4. Use of the primer-probe combination according to any one of claims 1 to 3 in preparing a product for detecting foodborne Clostridium, wherein the product for detecting foodborne Clostridium comprises a reagent, a kit, a chip, a test paper, a membrane strip or a detection platform. 5. A kit for detecting foodborne Clostridium, comprising the primer-probe combination according to any one of claims 1 to 3. 6. The kit according to claim 5, characterized in that the kit further comprises an enzyme mixture, a negative quality control product, a positive quality control product, and a PCR buffer, wherein the enzyme mixture contains at least one of a high-fidelity Taq enzyme, an UDG enzyme, a magnesium ion, and a dNTP. 7. The kit according to claim 6, characterized in that the negative quality control product is pure water treated with ethylene glycol pyrocarbonate.