Primer group and method for detecting listeria monocytogenes

Abstract

The invention relates to the technical field of biology, in particular to a primer group and a method for detecting listeria monocytogenes. The primer group for detecting the listeria monocytogenes, disclosed by the invention, comprises nucleotide sequences of primers as shown in SEQ ID NO. 1 to SEQ ID NO. 5; a milk sample can be directly used and added into an LAMP reaction reagent integrated in the micro-fluidic chip, the detection process is automatically completed by a full-automatic detection instrument, a spectrum is analyzed by a spectrum sensor, and a collected spectrum signal is comprehensively judged by an AI technology, so that full-automatic operation is realized, the detection time is controlled within one hour, and the detection accuracy is greatly improved. The system realizes real-time diagnosis, does not need professionals and laboratory environments, is portable and light, is low in price, does not need any complex operation, and can be operated and completed on site by detection personnel in a pasture only through simple training.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a primer set and method for detecting Listeria monocytogenes. Background Art

[0002] Listeria monocytogenes (LM) is an important pathogenic bacterium that causes listeriosis. Although this disease is not common, it can lead to serious health problems in specific populations. In particular, it can be fatal to pregnant women, newborns, the elderly, and immunocompromised individuals. Listeria monocytogenes is widely present in nature, including soil, water, and plants, and can even be found in some animals.

[0003] The main clinical manifestations of Listeria monocytogenes infection vary among individuals. In healthy adults, it may present as mild flu-like symptoms such as fever, muscle pain, nausea, vomiting, or diarrhea. However, in high-risk groups, it can cause more severe diseases, such as sepsis and meningitis. For pregnant women, it may lead to miscarriage, stillbirth, or severe neonatal infection. In addition, once infected, newborns may experience symptoms such as difficulty breathing, jaundice, and sepsis, and the disease progresses rapidly and critically. Currently, there is no specific vaccine for Listeria monocytogenes. Treatment mainly relies on antibiotic therapy, and especially ampicillin or penicillin G is often used as the first choice drug. For patients who are allergic to penicillin, other types of antibiotics can be considered.

[0004] In immunocompromised individuals, such as AIDS patients, cancer patients, or organ transplant recipients, listeriosis often presents in more complex and severe forms. In addition to common blood infections and meningitis, rare but fatal complications such as brainstem encephalitis may also occur. The condition of these patients is often more serious, and timely antibiotic treatment is required to avoid adverse consequences. In terms of the course of the disease, if not treated promptly and effectively, the condition may deteriorate rapidly, resulting in long-term hospitalization or even death. Therefore, for the above high-risk populations, preventive measures, early diagnosis, and treatment are particularly important.

[0005] The life cycle of Listeria monocytogenes mainly includes two aspects: reproduction in the host and transmission through the food chain. This bacterium can survive and reproduce in a low-temperature environment, which enables it to contaminate food even under refrigeration conditions, thus becoming a major hidden danger to public safety. Once it enters the human body, it can cross the intestinal wall into the bloodstream and further invade the central nervous system or other organ tissues, leading to corresponding pathological changes.

[0006] So far, the most common and convenient method for detecting the presence of LM is to perform ELISA (Enzyme-linked immunosorbent assay) after enrichment culture. This method is easy to operate and produces results quickly, but occasionally false positive or false negative results may occur, so further tests may be required to confirm the diagnosis. However, LM antibodies can also be detected in healthy populations, so this method has certain limitations. Enzyme-linked immunosorbent assay can also be used to judge by detecting antigen-antibody complexes in the body, but the positive rate of this detection method is only 67%, which undoubtedly greatly limits the detection of this virus.

[0007] Testing personnel can also use the molecular diagnostic technique - polymerase chain reaction (PCR) technique to diagnose LM in milk samples. This method is currently a relatively effective method with a relatively high detection rate of LM. Primers targeting the conserved region of the Listeria monocytogenes genome or primers designed based on the 18S rRNA nucleic acid sequence of Listeria monocytogenes are used to detect Listeria monocytogenes by the PCR method, and the results are specific and sensitive. Due to its high detection sensitivity, wide application range, and simple operation, the PCR technique is widely used. However, this detection technique has a complex procedure, requires precise instruments, and a long detection time, which is not conducive to on-site detection in non-laboratory environments and the popularization and application in grass-roots laboratories. Summary of the Invention

[0008] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a primer set and method for detecting Listeria monocytogenes.

[0009] On the one hand, the present invention provides a primer set for detecting Listeria monocytogenes, characterized in that the nucleotide sequences of the primers included in the primer set are as follows:

[0010] SEQ ID NO.1: 5’-ACTACTGAGCAAAAATCTTACG-3’;

[0011] SEQ ID NO.2: 5’-ATCCTAACTCCTGCATTGT-3’;

[0012] SEQ ID NO.3: 5’-GCTTCCCGTTAATCGAAAAATCATTAAAACACTTTTTCTATGTTTTCCAAACC-3’;

[0013] SEQ ID NO.4: 5’-TGGCTCTATTTGCGGTCAACAAAACAGTGTAATCTTGATGCCATC-3’;

[0014] SEQ ID NO.5: 5’-TCCTGACCTATGTGTATGGTAAAGA-3’。

[0015] Among them, the forward outer primer F3 has a nucleotide sequence as shown in SEQ ID NO.1; the reverse outer primer B3 has a nucleotide sequence as shown in SEQ ID NO.2; the forward inner primer FIP has a nucleotide sequence as shown in SEQ ID NO.3; the reverse inner primer BIP has a nucleotide sequence as shown in SEQ ID NO.4, and the loop primer LB has a nucleotide sequence as shown in SEQ ID NO.5.

[0016] Specifically, the present invention also provides the application of the above primer set. The above primer set can be applied to the preparation of a Listeria monocytogenes kit, or the application of detecting Listeria monocytogenes using the primer set, and the application of the primer set in a microfluidic chip for detecting Listeria monocytogenes, especially the detection of Listeria monocytogenes using a loop-mediated isothermal amplification - spectral sensor - artificial intelligence technology platform.

[0017] On the other hand, the present invention also provides a method for detecting Listeria monocytogenes, including: performing loop-mediated isothermal amplification on a sample to be tested using LAMP detection technology in combination with the above primer set; analyzing the amplification product.

[0018] Furthermore, the reaction temperature of the loop-mediated isothermal amplification is 65°C, and the reaction time is 35 minutes.

[0019] Furthermore, the reaction of the loop-mediated isothermal amplification is carried out in a microfluidic chip.

[0020] Furthermore, the method for analyzing the amplification product includes performing spectral analysis and / or colorimetric analysis on the LAMP reaction result.

[0021] The beneficial effect of the present invention is that, aiming at the LM-prfA gene sequence and LM-actA gene sequence of Listeria monocytogenes, the present invention has innovatively developed a set of specific primer formulations that do not require nucleic acid extraction and can directly use milk samples for LAMP reaction, and a method for rapid nucleic acid detection of Listeria monocytogenes by collecting data through a spectral sensor. This method can directly use milk samples and add them to the LAMP reaction reagents integrated in a microfluidic chip. The detection process is automatically completed by a fully automatic detection instrument, the spectrum is analyzed by a spectral sensor, and the collected spectral signals are comprehensively judged by AI technology to achieve full-automatic operation, control the detection time within one hour, achieve instant diagnosis, do not require professionals and a laboratory environment, are portable and lightweight, have a low price, do not require any complex operations, and can be completed by the detection personnel on the ranch through simple training.

[0022] Other features and advantages of the present invention will be set forth in the following description, and in part will be obvious from the description, or may be learned by practice of the present invention. The objectives and other advantages of the present invention are achieved and attained by the structure particularly pointed out in the description and the drawings.

[0023] In order to make the above objectives, features and advantages of the present invention more obvious and understandable, the following specifically enumerates preferred embodiments and, in conjunction with the accompanying drawings, the detailed description is as follows. Brief Description of the Drawings

[0024] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 is the screening result of the LAMP reaction of 4 groups of primer sets designed for the LM-prfA gene sequence and the LM-actA gene sequence of Listeria monocytogenes;

[0026] Figure 2 is the detection result of the present invention for positive and negative samples of Listeria monocytogenes;

[0027] Figure 3 is the detection result of the specificity experiment of the present invention;

[0028] Figure 4 is the detection result of the detection method for Listeria monocytogenes in milk samples of the present invention. Detailed Embodiments

[0029] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions of the present invention in conjunction with the drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0030] In the embodiments of the present invention, the LM standard positive samples are the synthesized LM-prfA gene sequence positive plasmid and the LM-actA gene sequence positive plasmid, Listeria welshimeri, Listeria ivanovii, Listeria seeligeri, and the primer sequences involved are all ordered from Sangon Biotech (Shanghai) Co., Ltd.

[0031] 1. Design and Screening of Primers

[0032] By using a higher concentration of LM standard positive samples, the following LAMP primer sets designed with LM-prfA nucleic acid fragments and LM-actA as templates were screened. The goal was to screen out a set of primers with the best sensitivity and specificity.

[0033] The primer design method is as follows:

[0034] (1) Download the Listeria monocytogenes prfA gene sequence (Sequence ID: LC006211.1) and actA gene sequence (Sequence ID: GU060678.1) from NCBI as primer design templates.

[0035] The Listeria monocytogenes prfA gene sequence (SEQ ID NO.6) is:

[0036]

[0037]

[0038] The Listeria monocytogenes actA gene sequence (SEQ ID NO.7) is:

[0039]

[0040]

[0041] (2) Use the software PrimerExplorer V5

[0042] (http: / / primerexplorer.jp / lampv5e / index.html), specific primers were designed using the Listeria monocytogenes - prfA gene sequence and actA gene sequence as templates. Based on six different regions labeled as F3, F2, F1, B1, B2, B3 in sequence from the 5' end on the Listeria monocytogenes prfA gene sequence and actA gene sequence, LAMP reaction primers were designed. Among them, F1, F2, F3, B1, B2, B3 are sequence fragments about 20bp long on the target gene, and F1c and B1c are the complementary regions of F1 and B1 respectively. The four primers include: upstream outer primer F3 (Forward Outer Primer), downstream outer primer B3 (Backward Outer Primer), upstream inner primer FIP (Forward Inner Primer), and downstream inner primer BIP (Backward Inner Primer). At the same time, using the free software PrimerExplorer V5, 1 - 2 loop primers LF (Loop Primer Forward) and LB (Loop Primer Backward) were designed between F1c and F2c, or between B1c and B2c to accelerate the LAMP reaction speed. The primers are required to meet the condition that the T m value (DNA strand melting temperature, melting temperature) of F1c and B1c is 64°C - 66°C, and the T m value of F2, F3, B2, B3 is 59°C - 61°C, and the T m value of the loop primers is 64°C - 66°C. The free energy at the 3' ends of F2 / B2, F3 / B3 and LF / LB and the 5' ends of F1c and B1c should not be greater than -4 kcal / mol. The free energy change (ΔG) is equal to the free energy of the product after the primer binds to the template minus the free energy of the initial reactants. The smaller ΔG (the larger the absolute value of the negative number), the easier the primer binds to the template. And the GC content in the primer sequence should be controlled at 45% - 65%. At the same time, ensure that each primer, especially the inner primer, is not prone to form a secondary structure to prevent the formation of primer dimers. There should be a certain appropriate spacing between the sequence fragments recognized by each primer. The spacing between the 5' end of F2 and the 5' end of B2 (LAMP amplification region) is 120 - 160 bases, the spacing between the 5' end of F2 and the 5' end of F1 (the region forming the loop structure) is 40 - 60 bases, and the interval between F2 and F3 is 0 - 60 bases. All the above conditions can be automatically calculated by the free software PrimerExplorer V5, and a list of the designed primers is given;

[0043] (3) Select multiple primer sets designed by the software PrimerExplorer V5 using the Listeria monocytogenes - prfA gene sequence and different positions on the actA gene as templates. By inputting the target product sequences of different primer sets into the NCBI Nucleotide BLAST

[0044] (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) online tool, analyze the specificity of the primer sets corresponding to the LAMP products to ensure that there is no non-specific amplification with other pathogens.

[0045] Through the above method, the sequences of 4 groups of LAMP primer sets were initially designed for selection. The nucleotide sequences of each group of primers are shown in Table 1 below:

[0046] Table 1 Primer Design

[0047]

[0048]

[0049] Using the above 4 groups of primer sets, with a high-concentration LM standard positive sample as the positive sample and sterilized deionized water (ddH2O) as the negative sample, perform the LAMP amplification experiment, and observe the color change of the LAMP reaction system of each primer set. As Figure 1 shown, the primer set of the 3rd group changed color (from purple-red to bright yellow) for the positive sample (+) in a shorter time, and the negative sample (-) did not change color at 45 min, indicating that this primer set has high sensitivity and specificity. However, the other primer sets screened in this round showed chaotic positive and negative reactions, so they were not selected; in summary, the 3rd group is the primer set with the best sensitivity and specificity. Therefore, the 3rd group of primer sets was selected as the primer set for detecting Listeria monocytogenes.

[0050] 2. Reaction Conditions

[0051] After several rounds of testing different reaction conditions, the following conditions were finally selected as the final reagent ratio and reaction conditions:

[0052] (1) Preparation of 10× primer mixture:

[0053] Table 2 Primer set mixed concentration ratio

[0054] Working concentration (μM) 10× Primer mix concentration (μM) FIP 1.6 16 BIP 1.6 16 F3 0.2 2 B3 0.2 2 LB 0.2 2

[0055] Table 3

[0056] Primer concentration (μM) Primer name For preparing 40 reactions (μL) 100 FIP 8 100 BIP 8 10 F3 10 10 B3 10 100 LB 2 Sterilized deionized water 12 Total volume (μL) 50

[0057] (2) Primer, 2×LAMP premix and positive / negative sample ratio in the LAMP reaction system:

[0058] Table 4

[0059] For 1 reaction (μL) 2× LAMP premix 6.25 10× Primer mix 1.25 Positive / negative sample 5.0

[0060] The 2×LAMP premix is a well-known formulation, containing MgSO4 (the concentration of MgSO4 is 6 mM to 12 mM), buffer, dNTPs, KCl, (NH4)2SO4, Bst DNA polymerase, phenol red, sterile enzyme-free water, etc.

[0061] The positive sample can be an LM standard positive sample, that is, the synthesized LM-prfA gene sequence positive plasmid and the LM-actA gene sequence positive plasmid.

[0062] The negative sample can be sterile deionized water (ddH2O).

[0063] (4) Reaction conditions

[0064] Under the temperature condition of 65 °C, constant temperature amplification for 35 minutes gives better detection results.

[0065] Heating can be carried out using a constant temperature heating device (water bath, metal bath, constant temperature oven, or PCR instrument, etc.).

[0066] 3. LAMP detection method for Listeria monocytogenes

[0067] The present invention uses an LM standard positive sample (that is, the synthesized LM-prfA gene sequence positive plasmid and the LM-actA gene sequence positive plasmid) and performs detection according to the following steps:

[0068] First step, preparation of Listeria monocytogenes positive / negative samples

[0069] Dilute the concentration of the LM standard positive sample to 1000 copies / μL as the positive sample; use sterile deionized water (ddH2O) as the negative sample.

[0070] Second step, prepare a 10× primer mixture according to the method in Table 3 above.

[0071] Third step, take the Listeria monocytogenes positive / negative samples prepared in the first step and prepare the LAMP reaction system for each reaction according to the method in Table 4 above.

[0072] Fourth step, heat at 65 °C for 35 minutes.

[0073] The reaction results are asFigure 2 As shown, the LAMP reaction systems of all 20 negative samples (-) were purplish red, and the LAMP reaction systems of all 20 positive samples (+) turned yellow. The positive accuracy rate in the reaction results was 100%, and the negative accuracy rate was 100%. It can be seen that Listeria monocytogenes can be clearly detected.

[0074] 4. Specificity experiment

[0075] To detect the specificity of the primer set for detecting Listeria monocytogenes, a mixture of Listeria welshimeri, Listeria ivanovii, and Listeria seeligeri was used as the cross-detection sample.

[0076] First step: Preparation of positive / negative samples of Listeria monocytogenes for cross-testing

[0077] Positive sample: The LM standard positive sample with a dilution concentration of 1000 copies / μL was used as the positive sample;

[0078] Negative sample: A mixture of Listeria welshimeri, Listeria ivanovii, and Listeria seeligeri with a concentration of 10 7 CFU / mL was used as the negative sample.

[0079] Second step: Prepare the 10× primer mixture according to the method in Table 3 above.

[0080] Third step: Respectively take the positive sample and negative sample prepared in the first step, and prepare the LAMP reaction systems for each reaction according to the method in Table 4 above.

[0081] Fourth step: Heat at 65°C for 35 minutes.

[0082] The reaction results are as Figure 3 shown. The LAMP reaction systems of the 2 positive samples (right) containing the synthetic LM-prfA gene sequence plasmid and LM-actA gene sequence plasmid solution both turned yellow, and the 5 negative samples (left) containing the mixture of Listeria welshimeri, Listeria ivanovii, and Listeria seeligeri did not change in color. It can be seen that no false positive results occurred, proving that the primer set for detecting Listeria monocytogenes of the present invention has good specificity.

[0083] According to Figure 2 and Figure 3 of the experimental results, the sensitivity and specificity of LAMP for detecting Listeria monocytogenes were preliminarily calculated. According to Figure 2 and Figure 3 of the experimental results, among the 22 positive samples, 22 showed clear positive results. Therefore, the sensitivity was calculated as: And all 25 of the 5 negative samples containing a mixture of Listeria welshimeri, Listeria ivanovii, and Listeria seeligeri and the 20 negative samples of sterilized deionized water (ddH2O) showed clear negative results. Therefore, the specificity was calculated as:

[0084]

[0085] 5. Loop-mediated isothermal amplification (LAMP) detection method for Listeria monocytogenes in milk samples

[0086] Step 1. Preparation of positive / negative milk samples

[0087] Negative milk samples: Dilute pure milk 100-fold with sterilized deionized water (ddH2O) to obtain negative milk samples;

[0088] Positive milk samples: Add a certain dilution ratio of LM standard positive samples (synthetic LM-prfA gene sequence positive plasmid solution with a concentration of 1000 copies / μL) to a portion of the above-prepared negative milk samples to obtain positive milk samples.

[0089] Step 2. Prepare a 10× primer mixture according to the method in Table 3 above.

[0090] Step 3. Prepare the LAMP reaction system for each reaction with the positive / negative milk samples prepared in Step 1 according to the method in Table 4 above.

[0091] Step 4. Heat at 65°C for 35 minutes.

[0092] The reaction results are as Figure 4 shown. All 10 negative milk samples (-) showed clear negative results, and all 10 positive milk samples (+) turned yellow. It can be seen that Listeria monocytogenes in milk samples can be clearly detected.

[0093] For the LAMP detection method of Listeria monocytogenes in milk samples, the sample treatment method is simple, nucleic acid extraction is not required, the detection steps are simplified, and the detection time is shortened. It only takes about 1 hour from sample collection to the detection result.

[0094] This detection method can also detect Listeria monocytogenes through the Loop-mediated Isothermal Amplification-Spectral Sensor-Artificial Intelligence (LAMP-SpectralSensor-AI) technology platform. Diluted milk samples can be directly injected into the LAMP reaction reagents integrated in the reaction chamber of the microfluidic chip for amplification. The amplification products are analyzed by a spectral sensor, and the collected spectral signals are comprehensively judged by AI technology, so as to quickly determine whether there is Listeria monocytogenes in the sample. Moreover, the spectral detection method combined with the AI algorithm can make the detection accuracy reach the molecular detection level. Cooperating with portable detection equipment, it can be seamlessly tested anytime and anywhere, realizing a convenient, fast, simple and easy-to-operate detection method with low cost. This technology requires no complex operations and can be completed by the detection personnel on the ranch through simple training on-site.

[0095] Inspired by the ideal embodiments of the present invention described above, through the above description, relevant staff can make various changes and modifications completely within the scope not deviating from the technical idea of this invention. The technical scope of this invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A primer set for detecting Listeria monocytogenes, characterized in that, The nucleotide sequences of the primers in the primer set are as follows: SEQ ID NO.1: 5’-ACTACTGAGCAAAAATCTTACG-3’; SEQ ID NO.2: 5’-ATCCTAACTCCTGCATTGT-3’; SEQ ID NO.3: 5’-GCTTCCCGTTAATCGAAAAATCATTAAAACACTTTTTCTATGTTTTCCAAACC-3’; SEQ ID NO.4: 5’-TGGCTCTATTTGCGGTCAACAAAACAGTGTAATCTTGATGCCATC-3’; SEQ ID NO.5: 5’-TCCTGACCTATGTGTATGGTAAAGA-3’.

2. Use of the primer set according to claim 1 in the preparation of a Listeria monocytogenes detection kit.

3. Use of the primer set according to claim 1 in the detection of Listeria monocytogenes.

4. Use of the primer set according to claim 1 in a microfluidic chip for detecting Listeria monocytogenes.

5. A detection method for Listeria monocytogenes, characterized in that, Comprising: Performing loop-mediated isothermal amplification on a sample to be tested by using the LAMP detection technique in combination with the primer set according to claim 1; Analyzing the result of the amplification product.

6. The detection method of Listeria monocytogenes according to claim 5, characterized in that the reaction temperature of the loop-mediated isothermal amplification is 65°C and the reaction time is 35 minutes.

7. The detection method of Listeria monocytogenes according to claim 5, characterized in that the reaction of the loop-mediated isothermal amplification is carried out in a microfluidic chip.

8. The detection method of Listeria monocytogenes according to claim 5, characterized in that the method for analyzing the result of the amplification product includes spectral analysis and / or colorimetric analysis of the LAMP reaction result.

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