CN112322763A

CN112322763A - LAMP primer for detecting escherichia coli F17 pilus adhesin gene and application thereof

Info

Publication number: CN112322763A
Application number: CN202011394942.4A
Authority: CN
Inventors: 陈颖钰; 郭爱珍; 陈鸣; 易萍; 陈曦; 孙亮; 张淏云; 朱杰; 胡长敏; 陈焕春
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-12-03
Filing date: 2020-12-03
Publication date: 2021-02-05

Abstract

The invention discloses a set of loop-mediated isothermal amplification (LAMP) primers for identifying a pilus adhesin gene of escherichia coli F17 and a fluorescent quantitative LAMP detection method established based on the primers. The primers comprise 2 outer primers, 2 inner primers and 2 loop primers, and the nucleotide sequence is shown as SEQ ID No. 2-7. The method is used for identifying the F17 antigen type of the Escherichia coli F17 adhesin gene, and has the advantages of high sensitivity, strong specificity, simple and quick operation and the like.

Description

LAMP primer for detecting escherichia coli F17 pilus adhesin gene and application thereof

Technical Field

The invention belongs to the field of animal molecular biology, and particularly relates to an LAMP primer for identifying a pilus adhesin gene of escherichia coli F17 and a fluorescent quantitative LAMP detection method.

Technical Field

Calf diarrhea is a common disease worldwide, and seriously affects the healthy development of the dairy and beef cattle breeding industries. The Escherichia coli F17 antigen type is firstly discovered by Girardeau and the like in foreigners in the attachment experiment of Escherichia coli separated from calf diarrhea, the Escherichia coli F17 pilus is one of common adhesins of bovine-derived enterotoxin-producing Escherichia coli (ETEC), and recent epidemiological data also show that the proportion of the Escherichia coli F17 antigen type in calf diarrhea-causing Escherichia coli is higher and higher, and huge economic loss is caused. Kolenda et al reviewed the study of virulence related factors of Escherichia coli in calves in 27 countries 106 papers between 1951 and 2013, and the detection rate of Escherichia coli F17 in 2328 calf diarrhea samples reaches 31.57%.

However, the current method for detecting the E.coli F17 pilus adhesin gene at home and abroad is mainly a PCR method, and more research reports show that the sensitivity of PCR has certain limitation. In the detection of Escherichia coli F17 antigen type, there is a possibility of detection omission. Therefore, a method with high sensitivity, simple and fast operation is urgently needed to make up for the defects of the existing detection method.

The loop-mediated isothermal amplification (LAMP) is a molecular biological detection method for completing DNA amplification by using the strand displacement activity of BstDNA polymerase at a constant temperature of 55-70 ℃. The traditional LAMP method has high sensitivity, but because the product is easy to generate aerosol pollution, false positive results often occur. The fluorescent quantitative LAMP method utilizes the principle of dye method qPCR, 1 mu L of Eva Green dye is added into the LAMP reaction system, and the amplification result can be observed and analyzed on a fluorescent quantitative PCR instrument; compared with the traditional LAMP method, the fluorescent quantitative LAMP method has the advantages that a tube cover does not need to be opened in the whole operation process, aerosol pollution risks are obviously reduced, relative quantitative analysis can be carried out through amplification time of positive templates with different concentrations, the reaction is rapid, the sensitivity is high, the pollution risk is low, and the like, and the fluorescent quantitative LAMP method has potential value for disease diagnosis.

Disclosure of Invention

The invention aims to design a set of specific LAMP primers for F17 pilus adhesin gene based on a fluorescence LAMP detection method, and establish a fluorescence quantitative LAMP detection method for identifying the Escherichia coli F17 pilus adhesin gene, which can be used for identifying the Escherichia coli F17 antigen type and improving the detection efficiency and sensitivity.

In order to achieve the aim of the invention, the invention provides a primer for fluorescent quantitative LAMP detection for identifying the Escherichia coli F17 pilus adhesin gene, and the nucleotide sequence of the primer is as follows:

an inner primer: FIP: 5'-TCATCGCGGTACCCCTGGAATAATTTATGGGCTGACGGAGGA-3' (SEQ ID No.2)

An inner primer: and (3) BIP: 5'-TGTCTTTTCCGGATGGTGCGTCCGGTCTTACGTGAACAGAC-3' (SEQ ID No.3)

An outer primer: f3: 5'-AGTCTTGACGGGCAGAGT-3' (SEQ ID No.4)

An outer primer: b3: 5'-CACTGTATTGCGCAGAGGAA-3' (SEQ ID No.5)

Loop primer: LF: 5'-CCCCATCCATATAAGGAGTCCC-3' (SEQ ID No.6)

Loop primer: LB: 5'-GGAAATTATGTATCAACGCAGGGAC-3' (SEQ ID No.7)

The primer is used for detecting the adhesion gene of the pilus of the escherichia coli F17, and the used detection method is a fluorescent quantitative LAMP method. The method takes Escherichia coli DNA as a template, recombinant plasmid pUC57-E.coli F17 constructed by Escherichia coli F17 pilin adhesion gene F17G (GenBank accession number: AF055311.1, SEQ ID No.1) as a positive control, takes sterile water as a negative control, takes inner primers shown by SEQ ID No.2 and SEQ ID No.3, outer primers shown by SEQ ID No.4 and SEQ ID No.5 and loop primers shown by SEQ ID No.6 and SEQ ID No.7 to perform LAMP amplification on Escherichia coli F17 pilin adhesion gene F17G, and collects fluorescent signals. If the sample to be detected and the positive standard substance can amplify a fluorescent signal, and the negative control amplification does not amplify a signal, the sample to be detected contains the F17 pilin adhesin gene, namely the Escherichia coli F17 antigen type, and if the sample to be detected and the negative control amplification does not amplify a fluorescent signal, and the positive standard substance can amplify a fluorescent signal, the sample to be detected does not contain the F17 pilin adhesin gene, namely the Escherichia coli F17 antigen type.

Coli F17 fimbriae are composed of two major proteins: structural pilus components and adhesins that recognize receptors. The pilus of the bacillus subtilis has four subtype structural subunits, so that the pilus is not suitable for being used as a detection target gene, the adhesion gene (F17G) is selected as the target gene in the research, the sequence has high specificity, and whether the infected escherichia coli is the escherichia coli F17 pilus adhesion gene or not can be judged by amplifying the sequence.

Wherein the reaction system of the fluorescence quantitative LAMP is as follows:

10×Isothermal Amplification Buffer 2.5μL

inner primer (40. mu. mol. L)^-1) FIP and BIP 1:1 mixed 2. mu.L

Outer primer (5. mu. mol. L)^-1) F3 and B31: 1 were mixed at 2. mu.L

Loop primer (10. mu. mol. L)^-1) LF and LB 1:1 were mixed in 2. mu.L

dNTPs MIX(10mM each) 2μL

Betaine (10 mol. L)^-1) 1μL

MgSO₄(100mM) 1μL

Bst DNA polymerase (8000U/mL) 1. mu.L

Eva Green 1μL

Template DNA 1. mu.L

Make up to 25 μ L with double distilled water.

The reaction temperature was 65 ℃.

The invention has the beneficial effects that:

the fluorescent quantitative LAMP method of the Escherichia coli F17 pilus adhesin gene is established by combining a fluorescent quantitative PCR technology with an LAMP technology, designing three pairs of primers according to the Escherichia coli F17 pilus adhesin gene F17G, adding Eva Green dye into the system, and collecting fluorescence through a fluorescent quantitative PCR instrument. Compared with TaqMan fluorescent quantitative PCR method, the LAMP reaction time is short, and the whole reaction can be completed within one hour. Compared with the common PCR, the LAMP reaction is sensitive, the cost is lower than that of a TaqMan fluorescence quantitative PCR method, the potential value of quickly and accurately detecting the Escherichia coli F17 antigen type is achieved, and the LAMP reaction has a good development prospect and a wide application space.

Drawings

FIG. 1: different temperatures are plotted against fluorescence signal intensity.

FIG. 2: different MgSO₄Concentration versus fluorescence signal intensity.

FIG. 3: graph of fluorescence signal versus betaine concentration.

FIG. 4: plot of fluorescence signal versus concentration of different dNTPs.

FIG. 5: graph of fluorescence signal versus concentration of different loop primers.

FIG. 6: graph of the relationship between plasmid concentration and fluorescence signal.

FIG. 7: different concentrations of plasmid PCR analysis diagram.

FIG. 8: graph showing the relationship between the E.coli liquid of type F17 and the fluorescence signal at different concentrations.

FIG. 9: PCR analysis of E.coli suspension F17 at different concentrations.

FIG. 10: and (5) fluorescence quantitative LAMP specificity detection result.

Detailed Description

The present invention will be described in detail below with reference to specific examples.

Example 1 establishment and optimization of detection methods

mu.L of DNA of recombinant plasmid pUC57-E.coli F17 inserted into E.coli F17 pilus adhesin gene F17G (GenBank accession No.: AF055311.1) was used as a template, and a reaction system was prepared as shown in Table 1, and sterile water was used as a negative control. When the concentration of one component is optimized, the other components are kept unchanged, and the volume of the modified components can be balanced by double distilled water.

TABLE 1 fluorescent quantitative LAMP reaction System (25. mu.L)

Component System (μ L)
	10×Isothermal Amplification Buffer 2.5
MgSO₄ 1.5
	dNTP Mix(10μM)3.5
FIP/BIP polymers (40. mu.M) 1 each
	F3/B3 Primers (5. mu.M) each 1
Loop F/Loop B prisms (10. mu.M) each 1
	Bst 2.0WarmStart DNA Polymerase(8000U/mL)1
Eva green 1
	DNA Sample 1
Nuclease-free Water 8.5

After the reaction solution is prepared, the reaction solution is centrifuged at the same time, the sample tube is placed into a C1000 Touch PCR instrument of Bio-Rad company, program setting and result analysis are carried out by using Bio-Rad CFX Manager software, and the LAMP reaction program is as follows: the cycle parameters of the sequence amplification of the F17 pilus adhesin gene F17G (GenBank accession number: AF055311.1) of Escherichia coli are as follows: 10s at 65 ℃ and 160 cycles were performed after the end of the second step at 65 ℃.

1.1 design of primers

A group of specific primers is designed by using online software PrimeExplorer V5, and the nucleotide sequence of the specific primers is as follows:

An outer primer: f3: 5'-AGTCTTGACGGGCAGAGT-3' (SEQ ID No.4)

An outer primer: b3: 5'-CACTGTATTGCGCAGAGGAA-3' (SEQ ID No.5)

Loop primer: LF: 5'-CCCCATCCATATAAGGAGTCCC-3' (SEQ ID No.6)

Loop primer: LB: 5'-GGAAATTATGTATCAACGCAGGGAC-3' (SEQ ID No.7)

1.2 recombinant plasmid construction

According to the sequence amplified by the primer, a recombinant plasmid pUC57-E.coli F17 is constructed, and a plasmid vector selects a commercial pUC57 plasmid, and the construction method is a conventional method in the field.

1.3 optimization of reaction conditions

1.3.1 temperature optimization

The LAMP reaction system is well mixed, then the mixture is respectively placed at the temperature of 59.9 ℃, 60.8 ℃, 62.5 ℃, 65 ℃, 68 ℃, 70.6 ℃, 72 ℃ and 72.7 ℃ for constant temperature reaction for 60 minutes, and the optimal reaction temperature is determined according to the fluorescence signal intensity and the Ct value after the reaction is finished.

The Ct values at different temperatures are shown in Table 2, the fluorescence intensities at different temperatures are shown in FIG. 1, and the optimum temperature is 65 ℃ according to the Ct values, i.e., the reaction time (Ct value 1 equals 22.5 seconds) and the different fluorescence signal intensities.

TABLE 2 different temperatures and corresponding Ct values

Temperature (. degree.C.)	72.7	72.0	70.6	68.0	65.0	62.5	60.8	59.9
									Ct value	N/A	N/A	85.20	67.79	52.90	59.50	68.29	77.81

1.3.2MgSO₄Concentration optimization

MgSO (MgSO)₄(100mM) was added to the PCR tube in an amount of 0. mu.L, 0.5. mu.L, 1. mu.L, 1.5. mu.L, 2. mu.L, 2.5. mu.L, 3. mu.L, respectively, and ddH was added thereto₂The amount of O is reduced accordingly, and the optimum MgSO is determined by repeating the experiment₄And (4) using the amount.

The Ct values at different dosages are shown in Table 3, the fluorescence intensities at different dosages are shown in FIG. 2, and the optimal dosage is 1 μ L, which is judged according to the Ct values, namely the reaction time (Ct value 1 equals 22.5 seconds) and the different fluorescence signal intensities.

TABLE 3 different MgSO₄Concentration and corresponding Ct value

MgSO₄(μL)	0.5	1.0	1.5	2.0	2.5	3.0
							Ct value	32.99	28.39	83.30	99.99	99.91	116.23

1.3.3 optimization of betaine dosage

Mixing betaine (10 mol. L)^-1) mu.L, 1. mu.L, 2. mu.L, 3. mu.L, 4. mu.L, 5. mu.L, 6. mu.L were added to the PCR tube, respectively, and ddH was added thereto₂The amount of O is reduced accordingly by repeatingThe optimum betaine dosage is determined by experiment.

The Ct values at different dosages are shown in Table 4, the fluorescence intensities at different dosages are shown in FIG. 3, and the optimal dosage is 1 μ L, which is judged according to the Ct values, namely the reaction time (Ct value 1 equals 22.5 seconds) and the different fluorescence signal intensities.

TABLE 4 different betaine concentrations and corresponding Ct values

1.3.4dNTPs dosage optimization

dNTPs (10mM) were added to the PCR tube in amounts of 1.5. mu.L, 2. mu.L, 2.5. mu.L, 3. mu.L, 3.5. mu.L, 4. mu.L, respectively, and ddH was added thereto₂The amount of O is reduced accordingly and the optimal dNTP dosage is determined by repeated experiments.

The Ct values at different dosages are shown in Table 5, the fluorescence intensities at different dosages are shown in FIG. 4, and the optimal dosage is 2 μ L, which is judged according to the Ct values, namely the reaction time (Ct value 1 equals 22.5 seconds) and the different fluorescence signal intensities.

TABLE 5 different dNTPs concentrations and corresponding Ct values

dNTPs(μL)	1.5	2	2.5	3	3.5	4
							Ct value	47.77	32.93	48.42	87.27	N/A	N/A

1.3.5 Loop primer concentration optimization

Fixing the concentration of the inner primer and the outer primer, increasing the concentration gradient of the loop primer, sequentially setting the concentration ratio of the outer primer to the loop primer according to 1:1, 1:2, 1:4, 1:6, 1:8 and 1:10, simultaneously setting a group of control groups without the loop primer, and using ddH₂O substitution, the optimal loop primer concentration was determined by repeated experiments.

Ct values at different concentrations are shown in Table 6, fluorescence intensities at different dosages are shown in FIG. 5, and the optimal concentration is 1:2 for the outer primer and the loop primer according to the Ct values, i.e., the reaction time (Ct value 1 equals 22.5 seconds) and different fluorescence signal intensities.

TABLE 6 concentration ratio of different outer primers to Loop primers and corresponding Ct value

An outer primer: loop primer

1:0

1:1

1:2

1:4

1:6

1:8

1:10

Ct value

N/A

88.50

80.83

84.32

90.14

90.84

88.88

In summary, the following steps: the invention designs a set of specific primers of F17 pilus adhesin gene, and establishes a method for fluorescence quantitative LAMP of Escherichia coli F17 pilus adhesin gene and F17 antigen type, wherein the optimal reaction system is as follows: to the reaction system were added 2.5. mu.L of 10 × Isotermal Amplification Buffer, 1. mu.L of MgSO₄(100mM), 1. mu.L betaine (10 mol. L-1), 2. mu.L dNTPs (10mM each), 2. mu.L inner primer (40. mu.M), 2. mu.L outer primer (5. mu.M), 2. mu.L loop primer (10. mu.M), 1. mu.L Bst 2.0 Watmstart DNA Polymerase (8000U/mL), 1. mu.L Eva green and 1. mu.L template DNA, and finally 9.5. mu.L ddH₂O to 25. mu.L, mixed well at 65 ℃ for 10s and 65 ℃ for 10s, and fluorescence collection is performed for 160 cycles after completion of the second step at 65 ℃.

Example 2 sensitive and specific detection

2.1 sensitivity assays

Plasmid of known concentration was diluted 10-fold (10)⁹-10⁰copies/. mu.L), LAMP and PCR experiments are carried out simultaneously, and the sensitivity is analyzed by comparing. LAMP resultsAs can be seen from the amplification curve of FIG. 6, the lowest detection limit was 10⁰copies/mu L, negative is not amplified, which indicates no pollution, and the experimental result has reliability. Table 7 shows that the Ct value of the plasmid was detected within 1h by LAMP detection. The PCR primers were the outer primers F3/B3 of the LAMP-specific primer set, and the results are shown in FIG. 7, with the lowest detection limit of 10³copies/. mu.L, negative without band, results with reliability. The LAMP sensitivity is 1000 times higher than that of PCR, which shows that the LAMP method of the invention has higher sensitivity.

TABLE 7 detection of plasmid sensitivity by LAMP method

Plasmid DNA copy number	10⁹	10⁸	10⁷	10⁶	10⁵
						Ct value	25.90	32.35	39.61	46.00	49.43
Plasmid DNA copy number	10⁴	10³	10²	10¹	10⁰
						Ct value	55.06	55.42	56.57	60.24	63.18

Escherichia coli F17 type bacterial suspension of known CFU was diluted 10-fold (3X 10)⁸-3 × 100CFU/mL), performing LAMP and PCR detection simultaneously after DNA extraction, and comparing analysis sensitivity. The LAMP result is shown in the amplification curve of FIG. 8, and the lowest detection limit is 3X 100CFU/mL, negative amplification is not generated, which indicates no pollution and reliable experimental results. Table 8 shows that the LAMP detection method detects Ct values of the bacterial content within 1 h. The PCR primers were the outer primers F3/B3 of the LAMP-specific primer set, and the results are shown in FIG. 9, with the lowest detection limit of 3X 10³CFU/mL, negative without band, results with reliability. The LAMP sensitivity is 1000 times higher than that of PCR, which shows that the LAMP method of the invention has higher analysis sensitivity.

TABLE 8 sensitivity of the LAMP method to the detection of bacterial solutions

Bacterial content (CFU/mL)	3×10⁸	3×10⁷	3×10⁶	3×10⁵	3×10⁴
						Ct value	49.44	57.41	67.18	74.1	87.13
Bacterial content (CFU/mL)	3×10³	10²	3×10¹	3×10⁰
						Ct value	90.73	96.72	100.23	135.51

2.2 specific detection

Simultaneously using 5 bacterial DNAs as templates, amplifying under the same conditions, and using ddH₂O as negative control, 1X 10⁵The copied recombinant plasmid pUC57-E.coli F17 is a positive template, and the specificity of the fluorescent quantitative LAMP method is identified. The 5 bacteria are respectively Escherichia coli K99 standard strain, Salmonella, Pasteurella, Streptococcus and GluconobacterStaphylococcus.

As shown in FIG. 10, only a single specific peak appears in the positive template, and no specific amplification products appear in the other five strains, which indicates that the method has good specificity and can be used for specific detection of the F17 pilus adhesin gene of Escherichia coli.

Sequence listing

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Claims

1. A set of LAMP primers for detecting a pilus adhesin gene of escherichia coli F17 is characterized by comprising 2 outer primers, 2 inner primers and 2 loop primers, wherein the nucleotide sequences of the outer primers, the inner primers and the loop primers are as follows:

an inner primer: FIP: 5'-TCATCGCGGTACCCCTGGAATAATTTATGGGCTGACGGAGGA-3'

An inner primer: and (3) BIP: 5'-TGTCTTTTCCGGATGGTGCGTCCGGTCTTACGTGAACAGAC-3'

An outer primer: f3: 5'-AGTCTTGACGGGCAGAGT-3'

An outer primer: b3: 5'-CACTGTATTGCGCAGAGGAA-3'

Loop primer: LF: 5'-CCCCATCCATATAAGGAGTCCC-3'

Loop primer: LB: 5'-GGAAATTATGTATCAACGCAGGGAC-3' are provided.

2. The LAMP primer of claim 1, which is used for detecting the F17 pilus adhesin gene of Escherichia coli.

3. A fluorescence LAMP method for detecting a pilus adhesin gene of Escherichia coli F17 is characterized by comprising the following steps: the method takes Escherichia coli DNA as a template, recombinant plasmids constructed by Escherichia coli F17 pilus adhesin genes as a positive control, and sterile water as a negative control, and the primers of claim 1 are used for LAMP amplification of the Escherichia coli F17 pilus adhesin genes to collect fluorescent signals; if the sample to be detected and the positive standard substance can amplify a fluorescence signal, and the negative control can not amplify the signal, the sample to be detected contains the Escherichia coli F17 pilin adhesin gene, and if the sample to be detected and the negative control can not amplify the fluorescence signal, and the positive standard substance can amplify the fluorescence signal, the sample to be detected does not contain the Escherichia coli F17 pilin adhesin gene.

4. The fluorescence LAMP method for detecting the F17 pilus adhesin gene of Escherichia coli as claimed in claim 3, wherein the LAMP amplification reaction system is as follows:

5. the fluorescence LAMP method for detecting the E.coli F17 pilus adhesin gene according to claim 3, wherein: the reaction temperature for the LAMP amplification was 65 ℃.