Target gene, primer group and detection method for cronobacter LAMP (loop-mediated isothermal amplification) detection

Abstract

The invention relates to the technical field of microbiological detection, and particularly discloses a target gene, a primer group and a detection method for cronobacter LAMP (Loop-Mediated Isothermal Amplification) detection. The target gene comprises one or more of ygcB, fimG, nanoK, papD, stD and phpB, 33 primers are designed according to the target gene, the primers are adopted by an LAMP detection method, a standard DNA sample with the concentration of 0.5 pg / mu l can be effectively detected, and the reaction time is only 30 min. The kit prepared by adopting the primers can realize rapid and efficient constant-temperature detection of Cronobacter spp., has low dependence on professional and complex experimental instruments, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and in particular relates to a target gene, a primer set and a detection method for Cronobacter LAMP detection. Background Art

[0002] Cronobacter belongs to the Enterobacteriaceae family. It is a Gram-negative, motile, facultative anaerobic conditional foodborne pathogen that is widely present in natural environments such as water and soil. The main route of infection is infant formula and infant complementary foods. The infection mainly occurs in newborns less than 4 weeks of age. It can cause neonatal necrotizing enterocolitis (NEC), bacteremia, and meningitis, with a mortality rate of up to 80%. It also causes severe neurological damage and other sequelae in survivors to a certain extent.

[0003] At present, the detection of Cronobacter in infant formula and baby food mainly uses the traditional selective culture method, which is time-consuming, labor-intensive, complicated to operate and has low detection efficiency. In recent years, emerging immunological detection methods such as enzyme-linked immunosorbent assay and immunofluorescence detection have the disadvantages of high cost, low precision, complex pretreatment and difficulty in automation. Molecular detection technologies such as PCR and qPCR require special laboratory equipment and professional experimenters, and are relatively time-consuming. Loop-mediated isothermal amplification (LAMP) is an emerging molecular biology technology developed in 2000 to achieve specific nucleic acid fragment amplification under constant temperature conditions. Its characteristics are that 4 specific primers are designed for 6 regions of the target gene, and the nucleic acid amplification reaction can be completed by amplifying under isothermal conditions (about 65°C) for 30-60 minutes using strand displacement DNA polymerase. The entire amplification process is simple, convenient, fast, low-cost and highly sensitive. It can amplify the amount of DNA to billions of times in a very short time (<1h), and has broad application prospects in the fields of animal and plant disease detection and food safety detection. Summary of the invention

[0004] The present invention provides a target gene, a primer set and a detection method for Cronobacter LAMP detection, which can realize rapid detection of Cronobacter and greatly shorten the detection cycle, and all operations can be completed within 1 hour.

[0005] The technical solution of the present invention is to provide a target gene for Cronobacter LAMP detection, wherein the target gene includes any one or a combination of the following genes: Selected from the genome of Cronobacter sakazakii ATCC 29544 yg Gene, DNA sequence is shown in SEQ ID NO.1; Selected from the genome of Cronobacter sakazakii ATCC 29544 fimG Gene and Kgene, and the DNA sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively; Selected from the genome of malonate-positive Cronobacter LMG23,826 papD Gene and sd gene, and the DNA sequences are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively; Selected from the genome of Cronobacter zurichensis z3032 phpB Gene, DNA sequence is shown as SEQ ID NO.6.

[0006] The present invention also relates to the application of the target gene in Cronobacter LAMP detection. yg Genes for detection of Cronobacter spp. fimG Gene and K gene, used to detect Cronobacter sakazakii; papD Gene and sd Gene for detection of malonate-positive Cronobacter ; phpB Genes for detection of Cronobacter zurichensis.

[0007] The present invention also relates to a primer set for Cronobacter LAMP detection, wherein the primer set is designed and prepared based on the DNA fragment sequence of the target gene, and the primer set is one or more sets, wherein one primer set at least comprises a forward outer primer, a forward inner primer, a reverse outer primer and a reverse inner primer, and further comprises 0 to 2 loop primers, wherein the primer sequences are shown in SEQ ID NOs. 7 to 39: The two external primers CLF3 and CLB3 and the two internal primers CLFIP and CLBIP corresponding to SEQ ID NO.1 have sequences shown in SEQ ID NOs.7 to 10, respectively; The two external primers SLF3 and SLB3, the two internal primers SLFIP and SLBIP, and the two loop guide primers SLLF and SLLB corresponding to SEQ ID NO.2 have sequences shown in SEQ ID NOs. 11 to 16, respectively; The two external primers SL-2F3 and SL-2B3, the two internal primers SL-2FIP and SL-2BIP, and the two loop guide primers SL-2LF and SL-2LB corresponding to SEQ ID NO.3 have sequences shown in SEQ ID NOs. 17 to 22, respectively; The two external primers MLF3 and MLB3, the two internal primers MLFIP and MLBIP, and the two loop guide primers MLLF and MLLB corresponding to SEQ ID NO. 4 have sequences shown in SEQ ID NOs. 23 to 28, respectively; The two external primers ML-2F3 and ML-2B3, the two internal primers ML-2FIP and ML-2BIP, and the two loop guide primers ML-2LF and ML-2LB corresponding to SEQ ID NO. 5 have sequences shown in SEQ ID NOs. 29 to 34, respectively; The two external primers TLF3 and TLB3, the two internal primers TLFIP and TLBIP, and the one loop guide primer TLLB corresponding to SEQ ID NO.6 have sequences shown in SEQ ID NOs.35-39, respectively.

[0008] The present invention also relates to the application of the primer set in the detection of Cronobacter LAMP, wherein the primers CLF3, CLB3, CLFIP and CLBIP are used to detect Cronobacter specific genes. yg ; Primers SLF3, SLB3, SLFIP, SLBIP, SLLF and SLLB were used to detect Cronobacter sakazakii specific genes fimG Primers SL-2F3, SL-2B3, SL-2FIP, SL-2BIP, SL-2LF, and SL-2LB were used to detect specific genes of Cronobacter sakazakii K ; Primers MLF3, MLB3, MLFIP, MLBIP, MLLF, and MLLB were used to detect malonate-positive Cronobacter specific genes papD Primers ML-2F3, ML-2B3, ML-2FIP, ML-2BIP, ML-2LF, and ML-2LB were used to detect malonate-positive Cronobacter specific genes sd ; Primers TLF3, TLB3, TLFIP, TLBIP and TLLB were used to detect Cronobacter zurichensis-specific genes phpB .

[0009] In a further preferred embodiment, the concentration of each primer in the primer set is 200 nM. The present invention also relates to a Cronobacter LAMP detection method, comprising the following steps: S1. Designing and preparing the primer set according to the target gene; S2. Prepare a LAMP detection system using the primer set of S1. The composition of the LAMP reaction mixture is as follows:

[0010] S3, prepare the genomic DNA of the sample to be tested, add it to the LAMP detection system in S2, and place it in a PCR instrument or a constant temperature water bath for circulation; S4. After amplification, place the positive samples under UV light for photographic analysis, or analyze the products by agarose gel electrophoresis.

[0011] In a further preferred embodiment, when preparing the primer set in S2, the target gene sequence is imported into the primer design webpage LAMP primer designing software PrimerExplorer, the parameters are set and the program is run to obtain 33 specific primer sequences SEQ ID NO.7~39 for the positive and reverse strands of Cronobacter and three important pathogenic bacteria.

[0012] In a further preferred embodiment, the cycle parameters in S3 are as follows: 65° C., 30 min.

[0013] The present invention also relates to the application of the detection method in the detection of food and environmental samples containing Cronobacter, the detection of clinical samples and / or epidemiological monitoring.

[0014] The present invention has the following beneficial effects: 1. The LAMP detection technology provided by the present invention can realize the rapid detection of Cronobacter and three important pathogenic species (Cronobacter sakazakii, malonate-positive Cronobacter and Cronobacter zurich), and can effectively detect a standard DNA sample with a concentration of 0.5 pg / μl, with a reaction time of only 30 minutes; the detection time can be shortened to within 1 hour throughout the whole process, and the purpose of rapid and efficient constant temperature detection of Cronobacter can be achieved. It has low dependence on professional and complex experimental instruments, has good market application prospects, and is of great significance for the clinical detection and epidemiological monitoring of important pathogenic bacteria of Cronobacter.

[0015] 2. The amplified product can be directly placed under UV light for photographing and analysis. The results are more intuitive and can be quickly observed and concluded. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the Cronobacter gene in Example 3 yg LAMP specific detection UV image and gel image.

[0017] Figure 2 is the Cronobacter sakazakii gene in Example 3 fimG LAMP specific detection UV image and gel image.

[0018] Figure 3 is the Cronobacter sakazakii gene in Example 3 K LAMP specific detection UV image and gel image.

[0019] Figure 4 is the malonate-positive Cronobacter gene in Example 3 papD LAMP specific detection UV image and gel image.

[0020] Figure 5 is the malonate-positive Cronobacter gene in Example 3 sd LAMP specific detection UV image and gel image.

[0021] Figure 6 is the Cronobacter zurich gene in Example 3 phpB LAMP specific detection UV image and gel image.

[0022] Figure 7 These are the UV and gel images of the sensitivity detection of Cronobacter sakazakii in Example 4.

[0023] Figure 8 These are the UV and gel images of the sensitivity detection of malonate-positive Cronobacter in Example 4.

[0024] Fig. 9 These are the UV images and gel images of the sensitivity detection of Cronobacter Zurich in Example 4. DETAILED DESCRIPTION

[0025] The present invention is described in detail below in conjunction with specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and a specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0026] Example 1 1. Selected from Cronobacter sakazakii ATCC 29544 yg , fimG and K As the target gene, malonate-positive Cronobacter LMG23,826 was selected papD and sd The target gene was selected from Cronobacter zurich z3032 phpB is the target gene. The gene sequence is shown in SEQ ID NO.1~6.

[0027] 2. Import the selected target gene sequences for Cronobacter genus and species into the primer design webpage LAMPprimer designing software PrimerExplorer and set the parameters. Run the program to obtain 33 specific primer sequences for the forward and reverse strands of Cronobacter genus and three important pathogenic bacteria, as shown in Table 1 below.

[0028] Table 1

[0029] 3. Send the designed primer sequences to Sangon Biotech Co., Ltd. for DNA synthesis and purification by PAGE for later use.

[0030] Example 2: Establishment of LAMP for Cronobacter and Important Pathogenic Species 1. First, use the bacterial genomic DNA extraction kit (product number DP302-02) of Tiangen Biochemical Technology (Beijing) Co., Ltd. and follow its operating steps to extract bacterial genomic DNA.

[0031] 2. Take 1 μl of the above genomic DNA extract as a template and add it to the LAMP reaction mixture. The composition of the LAMP reaction mixture is shown in Table 2.

[0032] Table 2. LAMP reaction mixture composition

[0033] 3. Place the reaction mixture obtained in step 2 in a PCR instrument (applied biosystems) or a constant temperature water bath, set the cycle parameters and run. The cycle parameters in this embodiment are: constant temperature water bath at 65°C for 30 minutes.

[0034] Embodiment 3, The method established in Example 2 was used to perform LAMP specific detection on new specific genes of Cronobacter and three important pathogenic species.

[0035] 1. New specific genes of Cronobacter yg LAMP specific detection UV image and gel image are shown in Figure 1 Lane M: DNA Marker 2000; Lane 1: Cronobacter sakazakii G4023; Lane 2: Malonate-positive Cronobacter G3864; Lane 3: Cronobacter Zurich G3874; Lane 4: Cronobacter Dublin G4040; Lane 5: Cronobacter univ. G3876; Lane 6: Enterobacter cloacae; Lane C: Negative control. Figure 1 It can be seen that Cronobacter sakazakii, malonate-positive Cronobacter, Cronobacter Zurich, Cronobacter Dublin, and Cronobacter univesus were all specifically amplified. 2. New specific genes of Cronobacter sakazakii fimG LAMP specific detection UV image and gel image are shown in Figure 2 Lane M: DNA Marker 2000; Lane 1: Cronobacter sakazakii G4023; Lane 2: Malonate-positive Cronobacter G3864; Lane 3: Cronobacter Zurich G3874; Lane 4: Cronobacter Dublin G4040; Lane 5: Cronobacter univ. G3876; Lane 6: Enterobacter cloacae; Lane C: Negative control. Figure 2 It can be seen that Cronobacter sakazakii was specifically amplified.

[0036] 3. New specific genes of Cronobacter sakazakii K LAMP specific detection UV image and gel image are shown in Figure 3 Lane M: DNA Marker 2000; Lane 1: Cronobacter sakazakii G4023; Lane 2: Malonate-positive Cronobacter G3864; Lane 3: Cronobacter Zurich G3874; Lane 4: Cronobacter Dublin G4040; Lane 5: Cronobacter univ. G3876; Lane 6: Enterobacter cloacae; Lane C: Negative control. Figure 3 It can be seen that Cronobacter sakazakii was specifically amplified.

[0037] 4. New specific genes of malonate-positive Cronobacter papD LAMP specific detection UV image and gel image are shown in Figure 4 Lane M: DNA Marker 2000; Lane 1: Cronobacter sakazakii G4023; Lane 2: Malonate-positive Cronobacter G3864; Lane 3: Cronobacter Zurich G3874; Lane 4: Cronobacter Dublin G4040; Lane 5: Cronobacter univ. G3876; Lane 6: Enterobacter cloacae; Lane C: Negative control. Figure 4 It can be seen that malonate-positive Cronobacter was specifically amplified. 5. New specific genes of malonate-positive Cronobacter sd LAMP specific detection UV image and gel image are shown in Figure 5 Lane M: DNA Marker 2000; Lane 1: Cronobacter sakazakii G4023; Lane 2: Malonate-positive Cronobacter G3864; Lane 3: Cronobacter Zurich G3874; Lane 4: Cronobacter Dublin G4040; Lane 5: Cronobacter univ. G3876; Lane 6: Enterobacter cloacae; Lane C: Negative control. Figure 5 It can be seen that malonate-positive Cronobacter was specifically amplified. 6. New specific genes of Cronobacter zurichensis phpB LAMP specific detection UV image and gel image are shown in Figure 6Lane M: DNA Marker 2000; Lane 1: Cronobacter sakazakii G4023; Lane 2: Malonate-positive Cronobacter G3864; Lane 3: Cronobacter Zurich G3874; Lane 4: Cronobacter Dublin G4040; Lane 5: Cronobacter univ. G3876; Lane 6: Enterobacter cloacae; Lane C: Negative control. Figure 6 It can be seen that Cronobacter zurichensis was specifically amplified.

[0038] Example 4 The method established in Example 2 was used to detect the sensitivity of the new specific gene LAMP of Cronobacter and three important pathogenic species, and samples of different concentrations were taken for testing. The specific results are as follows: 1. UV and gel images of sensitivity test of Cronobacter sakazakii G4023 are shown in Figure 7 Lane M: DNA Marker 2000; Lane 1: 50 ng / μl; Lane 2: 5 ng / μl; Lane 3: 50 pg / μl; Lane 4: 5 pg / μl; Lane 5: 0.5 pg / μl; Lane 6: 0.05 pg / μl; Enterobacter cloacae; Lane C: negative control. Figure 7 It can be seen that the minimum detection sensitivity of Cronobacter sakazakii is 0.5 pg / μl.

[0039] 2. Malonate-positive Cronobacter G3864 sensitivity test UV graph and gel graph are shown in Figure 8 Lane M: DNAMarker 2000; Lane 1: 50 ng / μl; Lane 2: 5 ng / μl; Lane 3: 50 pg / μl; Lane 4: 5 pg / μl; Lane 5: 0.5 pg / μl; Lane 6: 0.05 pg / μl; Enterobacter cloacae; Lane C: negative control. Figure 8 It can be seen that the minimum detection sensitivity of malonate-positive Cronobacter is 0.5 pg / μl.

[0040] 3. UV and gel images of sensitivity test of Zurich Cronobacter G3874 are shown in Figure 8 Lane M: DNAMarker 2000; Lane 1: 50 ng / μl; Lane 2: 5 ng / μl; Lane 3: 50 pg / μl; Lane 4: 5 pg / μl; Lane 5: 0.5 pg / μl; Lane 6: 0.05 pg / μl; Enterobacter cloacae; Lane C: negative control. Figure 8 It can be seen that the minimum detection sensitivity of Cronobacter Zurich is 0.5 pg / μl.

[0041] The above embodiments describe the preferred implementation of the present invention, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A target gene for Cronobacter LAMP detection, characterized in that: The target gene includes any one or more of the following genes: Selected from the genome of Cronobacter sakazakii ATCC 29544 yg Gene, DNA sequence is shown in SEQ ID NO.1; Selected from the genome of Cronobacter sakazakii ATCC 29544 fimG Gene and K gene, and the DNA sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively; Selected from the genome of malonate-positive Cronobacter LMG23826 papD Gene and sd gene, and the DNA sequences are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively; Selected from the genome of Cronobacter zurichensis z3032 phpB Gene, DNA sequence is shown as SEQ ID NO.

6.

2. The use of the target gene according to claim 1 in the detection of Cronobacter LAMP, characterized in that: yg Genes for detection of Cronobacter spp. fimG Gene and K gene, used to detect Cronobacter sakazakii; papD Gene and sd Gene for detection of malonate-positive Cronobacter ; phpB Genes for detection of Cronobacter zurichensis.

3. A primer set for LAMP detection of Cronobacter, characterized in that: The primer set is designed and prepared according to the DNA fragment sequence of the target gene according to claim 1, and the primer set is one or more sets, and one primer set at least comprises a forward outer primer, a forward inner primer, a reverse outer primer and a reverse inner primer, and also comprises 0 to 2 loop primers, wherein the primer sequences are shown in SEQ ID NO.7 to 39: The two external primers CLF3 and CLB3 and the two internal primers CLFIP and CLBIP corresponding to SEQ ID NO.1 have sequences shown in SEQ ID NOs.7 to 10, respectively; The two external primers SLF3 and SLB3, the two internal primers SLFIP and SLBIP, and the two loop guide primers SLLF and SLLB corresponding to SEQ ID NO.2 have sequences shown in SEQ ID NOs. 11 to 16, respectively; The two external primers SL-2F3 and SL-2B3, the two internal primers SL-2FIP and SL-2BIP, and the two loop guide primers SL-2LF and SL-2LB corresponding to SEQ ID NO.3 have sequences shown in SEQ ID NOs. 17 to 22, respectively; The two external primers MLF3 and MLB3, the two internal primers MLFIP and MLBIP, and the two loop guide primers MLLF and MLLB corresponding to SEQ ID NO. 4 have sequences shown in SEQ ID NOs. 23 to 28, respectively; The two external primers ML-2F3 and ML-2B3, the two internal primers ML-2FIP and ML-2BIP, and the two loop guide primers ML-2LF and ML-2LB corresponding to SEQ ID NO. 5 have sequences shown in SEQ ID NOs. 29 to 34, respectively; The two external primers TLF3 and TLB3, the two internal primers TLFIP and TLBIP, and the one loop guide primer TLLB corresponding to SEQ ID NO.6 have sequences shown in SEQ ID NOs.35-39, respectively.

4. Use of the primer set according to claim 3 in LAMP detection of Cronobacter, characterized in that: Primer set consisting of primers CLF3, CLB3, CLFIP and CLBIP is used to detect Cronobacter specific genes yg ; The primer set consisting of primers SLF3, SLB3, SLFIP, SLBIP, SLLF and SLLLB is used to detect the specific genes of Cronobacter sakazakii fimG The primer set consisting of primers SL-2F3, SL-2B3, SL-2FIP, SL-2BIP, SL-2LF and SL-2LB is used to detect the specific gene of Cronobacter sakazakii K ; Primer set consisting of primers MLF3, MLB3, MLFIP, MLBIP, MLLF and MLLB is used to detect malonate-positive Cronobacter specific genes papD The primer set consisting of primers ML-2F3, ML-2B3, ML-2FIP, ML-2BIP, ML-2LF and ML-2LB is used to detect the specific gene of malonate-positive Cronobacter sd ; Primer set consisting of primers TLF3, TLB3, TLFIP, TLBIP and TLLB is used to detect specific genes of Cronobacter zurichensis phpB .

5. The use according to claim 4, characterized in that: The concentration of each primer in the primer set was 200 nM.

6. A Cronobacter LAMP detection method, characterized in that: The following steps are involved: S1. Design the target gene according to claim 1 or 2 and prepare the primer set according to any one of claims 3 to 5; S2. Prepare a LAMP detection system using the primer set of S1. The composition of the LAMP reaction mixture is as follows: S3, prepare the genomic DNA of the sample to be tested, add it to the LAMP detection system in S2, and place it in a PCR instrument or a constant temperature water bath for circulation; S4. After amplification, place the positive samples under UV light for photographic analysis, or analyze the products by agarose gel electrophoresis.

7. The detection method according to claim 6, characterized in that: When preparing the primer set in S2, the target gene sequence was imported into the primer design webpage LAMP primer designing software PrimerExplorer, the parameters were set and the program was run to obtain 33 specific primer sequences SEQ ID NO.7~39 for the positive and reverse strands of Cronobacter and three important pathogenic bacteria.

8. The detection method according to claim 6, characterized in that: The cycle parameters in S3 were as follows: 65°C, 30 min.

9. Use of the detection method according to any one of claims 6 to 8 in the detection of food, environmental samples, clinical samples and / or epidemiological monitoring containing Cronobacter.

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