CN113789397A - Composition, kit and detection method for detecting vibrio cholerae O1 group and O139 group - Google Patents
Composition, kit and detection method for detecting vibrio cholerae O1 group and O139 group Download PDFInfo
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Abstract
The invention belongs to the field of molecular biological detection, and discloses a composition, a kit and a detection method for detecting vibrio cholerae O1 group and O139 group. The composition comprises: the nucleotide sequence of the primer pair 1 for detecting the vibrio cholerae O1 group is shown as SEQ ID NO: 1-2; the nucleotide sequence of the primer pair 2 for detecting the vibrio cholerae O139 group is shown as SEQ ID NO: 3-4. The composition may further comprise: the probe A for detecting the vibrio cholerae O1 group has a nucleotide sequence shown as SEQ ID NO: 5 is shown in the specification; the nucleotide sequence of the probe B for detecting the vibrio cholerae O139 group is shown as SEQ ID NO: and 6. The detection method has the characteristics of good specificity, high sensitivity, simplicity, convenience, rapidness and practicability, and can meet the detection requirements of vibrio cholerae in field and field environments.
Description
Technical Field
The invention belongs to the field of molecular biological detection, and particularly relates to a composition, a kit and a detection method for detecting vibrio cholerae O1 group and O139 group.
Background
Cholera (Cholera) is an ancient, virulent intestinal infectious disease, known as "one of the most terrible pestilences of the earth". The clinical manifestations of the disease are acute diarrhea and vomiting, frequent diarrhea and vomiting and large quantity, and the disease is a rice swill water sample, thereby causing dehydration, electrolyte disorder and peripheral circulatory failure, and severe patients can cause shock and acidosis, and if the patients are not rescued in time, the disease death rate is very high. Patients with mild symptoms sometimes resemble enteritis, are easy to miss diagnosis or misdiagnose, and have the same non-negligible harmfulness. The gram-negative bacterium, Vibrio Cholerae (VC), is a pathogenic bacterium responsible for cholera.
Vibrio cholerae is classified as Vibrio of the family Vibrionaceae. According to the difference of thallus (O) antigens, the vibrio cholerae can be divided into a plurality of O serogroups. More than 200O serogroups (O serogroups) have been identified, but only the O1 and O139 groups of Vibrio cholerae have been found to induce cholera, and serogroups other than the O1 and O139 groups are collectively referred to as non-O1/O139 groups.
As cholera usually comes rapidly and can be spread rapidly through water sources, etc., the cholera is easy to cause world pandemics and is determined as an international quarantine infectious disease. China stipulates that cholera belongs to class A infectious diseases in infectious disease prevention and treatment laws, and important monitoring and quarantine are necessary. Therefore, it is necessary to establish a nucleic acid detection method for pathogenic serogroups (O1 group and O139 group) of vibrio cholerae, which can be applied to the field or even the field, and has important significance for pathogen detection and improvement of epidemic situation response capability.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a composition, a kit and a detection method for detecting vibrio cholerae O1 group and O139 group. The detection method has the characteristics of good specificity, high sensitivity, simplicity, convenience, rapidness and practicability, and can meet the detection requirements of vibrio cholerae in field and field environments.
The invention provides a composition for detecting vibrio cholerae O1 group and O139 group, which comprises the following components in part by weight:
the primer pair 1 for detecting the vibrio cholerae O1 group has the nucleotide sequence as follows:
a forward primer: 5'-CATTCACTTATGTTGCCTCGGT-3' (SEQ ID NO: 1),
reverse primer: 5'-GACTCACCTTCGATTTCAGCA-3' (SEQ ID NO: 2);
and a primer pair 2 for detecting vibrio cholerae O139 group, wherein the nucleotide sequence is as follows:
a forward primer: 5'-GTTCCCTTGTTAGACCACCG-3' (SEQ ID NO: 3),
reverse primer: 5'-CCTTTCCACCTCGGTATTTCA-3' (SEQ ID NO: 4).
The composition for detecting the vibrio cholerae O1 group and the vibrio cholerae O139 group is used for detecting an object to be detected, so that the simultaneous detection of the vibrio cholerae O1 group and the vibrio cholerae O139 group can be realized, and specific pathogenic vibrio cholerae serogroups can be distinguished.
Preferably, the composition further comprises:
the probe A for detecting the vibrio cholerae O1 group has the nucleotide sequence as follows: 5'-AATACCATAGTCCAGTGTG-3' (SEQ ID NO: 5);
and a probe B for detecting vibrio cholerae O139 group, wherein the nucleotide sequence of the probe B is as follows: 5'-TGCTGAGTTTGCTGCCAGTT-3' (SEQ ID NO: 6).
More preferably, the 5 'ends of the probe A and the probe B are both labeled with a fluorescent group, and the 3' ends of the probe A and the probe B are both labeled with a quenching group; and the fluorescent groups marked by the probe A and the probe B are different.
Wherein the fluorescent group includes, but is not limited to, the following types: FAM, CY5, TEXAS RED, Tet, HEX; the quencher groups include, but are not limited to, the following types: BHQ1, BHQ2, TAMRA, ECLIPSE, MGB.
Further preferably, the fluorescent group labeled by the probe a is FAM, and the fluorescent group labeled by the probe B is HEX. And the quenching groups marked by the probe A and the probe B are MGBs.
The invention also provides a kit for detecting the vibrio cholerae O1 group and O139 group, which comprises the composition.
Preferably, the kit further comprises four nucleotides, a positive quality control substance, a negative quality control substance, a magnesium ion buffer, formamide, ammonium sulfate, trehalose and Taq DNA polymerase, wherein the four nucleotides are dATP, dGTP, dCTP and dUTP.
More preferably, the negative quality control substance is TE buffer solution or ultrapure water. The TE buffer solution is prepared from Tris and EDTA, and the prepared water is ultrapure water without ribonuclease.
More preferably, the positive quality control substance is a substance containing a nucleotide sequence shown as SEQ ID NO:7 in the presence of a plasmid pGEM-T. The nucleotide sequence shown as SEQ ID NO.7 comprises specific conserved sequences of vibrio cholerae O1 group and O139 group.
TGTACCAACATTCACTTATGTTGCCTCGGTTAATACCATAGTCCAGTGTGGTGCGTTACCCGTTTTTGCTGAAATCGAAGGTGAGTCTCTACAAGTGAGCTTCATTAGAAGGGCGGGTTCCCTTGTTAGACCACCGCATTGCTGAGTTTGCTGCCAGTTTGCCGATCCATTTGAAATACCGAGGTGGAAAGGGAAAGTGG(SEQ ID NO.7)。
The invention also provides a detection method of the vibrio cholerae O1 group and O139 group, which comprises the following steps:
and extracting DNA of a sample to be detected, and detecting the DNA by adopting the kit.
Specifically, the DNA of a sample to be detected is extracted, the DNA is added into a thermal convection PCR tube, and the kit and the thermal convection PCR detection equipment are adopted for detection.
The invention adopts a constant temperature thermal isolation type PCR (iPCR) method to detect vibrio cholerae, the constant temperature thermal isolation type PCR (iPCR) is also called as thermal convection PCR, and is developed by a novel simple PCR amplification technology established by Krishnan M and the like in 2002. The iPCR utilizes the principle of thermal convection of the solution, i.e., when heating the bottom of a container containing liquid, the liquid moves downward due to gravity because of the cooler upper surface and higher specific gravity; the liquid heated at the lower part is extruded to move upwards because of the lower specific gravity, and therefore, the circulation of the liquid is naturally formed. This cycle continues as the warmer liquid flows to the surface, cooling due to heat dissipation from the environment, and the cooler liquid flows to the bottom, heating due to heat, thus creating a steady temperature gradient. If the shape and material of the heated container are specially designed, the optimal pipe diameter and height are found, for example, the R-Tube PCR Tube with the lower part being thin and the upper part being thick on the market can skillfully control the convection time and the heat dissipation rate, and a local environment suitable for the denaturation-annealing-extension and other reactions of PCR can be formed.
Preferably, in the thermal convection PCR tube, the final concentration of each primer is 0.3-0.8. mu. mol/L, and the final concentration of each probe is 0.2-0.4. mu. mol/L.
Preferably, the reaction procedure of the detection method is as follows: keeping the temperature at 50 ℃ for 8-10 min; and carrying out thermal convection PCR reaction at 95 ℃ for 45-50 min.
Preferably, the thermal convection PCR detection device is POCKITTMOn-site nucleic acid detection equipment (taiwan ruiji ocean biotechnology limited). After the reaction is finished, the pockitt device can automatically convert the signal-to-noise ratio (S/N) of the fluorescence signal change into 3 types of positive, negative or questionable results according to a built-in algorithm and a default S/N threshold, which are respectively expressed as "+", "-" and "? "is displayed on the touch screen of the instrument. "+" indicates a positive result, "-" indicates a negative result, "? "indicates that the result is ambiguous and needs to be retested.
Compared with the prior art, the invention has the following beneficial effects:
(1) the specific primers and the probes designed by the invention can be arranged in the same PCR system, can realize the detection and the differentiation of vibrio cholerae O1 group and O139 group at the same time, and have good specificity and sensitivity;
(2) the invention adopts the thermal convection PCR method to detect the vibrio cholerae, has the advantages of simple and convenient operation, practicability and low cost, and the detection instrument is easy to carry and is suitable for quick detection in the field or even in the field; in addition, a cover does not need to be opened in the detection process, and the sample can be effectively prevented from being polluted;
(3) the detection method is short in time, and the detection result can be obtained by detecting on a computer for 50-60 min.
Drawings
FIG. 1 shows the results of detection of Vibrio cholerae of non-O1/O139 group in a specificity test;
FIG. 2 shows the results of detection of Vibrio cholerae group O1 in a specificity test;
FIG. 3 shows the results of detection of Vibrio cholerae of O139 group in a specificity test.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are only preferred embodiments of the present invention, and the claimed protection scope is not limited thereto, and any modification, substitution, combination made without departing from the spirit and principle of the present invention are included in the protection scope of the present invention.
The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.
Example 1
The present embodiment provides a composition for detecting vibrio cholerae group O1 and O139, comprising:
the primer pair 1 for detecting the vibrio cholerae O1 group has the nucleotide sequence as follows:
a forward primer: 5'-CATTCACTTATGTTGCCTCGGT-3' (SEQ ID NO: 1),
reverse primer: 5'-GACTCACCTTCGATTTCAGCA-3' (SEQ ID NO: 2);
the primer pair 2 for detecting the vibrio cholerae O139 group has the nucleotide sequence as follows:
a forward primer: 5'-GTTCCCTTGTTAGACCACCG-3' (SEQ ID NO: 3),
reverse primer: 5'-CCTTTCCACCTCGGTATTTCA-3' (SEQ ID NO: 4);
the probe A for detecting the vibrio cholerae O1 group has the nucleotide sequence as follows: 5'-AATACCATAGTCCAGTGTG-3' (SEQ ID NO: 5);
the probe B for detecting the vibrio cholerae O139 group has the nucleotide sequence as follows: 5'-TGCTGAGTTTGCTGCCAGTT-3' (SEQ ID NO: 6).
Wherein the 5 'end of the probe A for detecting the vibrio cholerae O1 group is marked with a fluorescent group FAM, and the 3' end is marked with a quenching group MGB. The 5 'end of the probe B for detecting the vibrio cholerae O139 group is marked with a fluorescent group HEX, and the 3' end is marked with a quenching group MGB.
Example 2
This example provides a kit for detecting vibrio cholerae O1 and O139, comprising the composition of example 1 (including primer pair 1, primer pair 2, probe a, and probe B), and further comprising four nucleotides dATP, dGTP, dCTP, and dUTP, a positive quality control substance, a negative quality control substance, a magnesium ion buffer, formamide, ammonium sulfate, trehalose, and Taq DNA polymerase.
The negative quality control substance is TE buffer solution, the TE buffer solution is prepared from Tris and EDTA, and the prepared water is ultrapure water without ribonuclease.
Wherein the positive quality control substance is a substance containing the amino acid sequence shown as SEQ ID NO:7 in the presence of a plasmid pGEM-T.
TGTACCAACATTCACTTATGTTGCCTCGGTTAATACCATAGTCCAGTGTGGTGCGTTACCCGTTTTTGCTGAAATCGAAGGTGAGTCTCTACAAGTGAGCTTCATTAGAAGGGCGGGTTCCCTTGTTAGACCACCGCATTGCTGAGTTTGCTGCCAGTTTGCCGATCCATTTGAAATACCGAGGTGGAAAGGGAAAGTGG(SEQ ID NO.7)。
Example 3
The present embodiment provides a method for detecting vibrio cholerae group O1 and group O139, comprising the following steps:
DNA of a sample to be tested was extracted, and 5. mu.L of the extracted DNA, 2.5. mu.L of Taq DNA polymerase and 17.5. mu.L of ultrapure water were added to a PCR Tube (R-Tube) for thermal convection to obtain 25. mu.L of a reaction mixture. The reaction mixture also included four nucleotides, dATP, dGTP, dCTP and dUTP, the composition of example 1 (including primer pair 1, primer pair 2, probe a and probe B), as well as magnesium buffer, formamide, ammonium sulfate and trehalose. The final concentration of each primer was 0.6. mu. mol/L and the final concentration of the probe was 0.3. mu. mol/L.
Sealing cap with R-Tube reaction Tube, centrifuging in special centrifuge, and placing in POCKITTMDetection was performed in a nucleic acid analyzer (taiwan ruiji ocean biotechnology limited).
The reaction program for the iPCR detection is as follows:
the dual wavelength mode of "520 nm and 550 nm" is selected, and the detection time is the default 58min of the system in the instrument. The built-in program is set as follows: first, 50 ℃ incubation was performed for 10min, and then 95 ℃ thermal convection PCR reaction was performed for about 48 min. The detection results of the vibrio cholerae O1 group and the vibrio cholerae O139 group are obtained through instrument display, wherein the wavelength of 520nm is shown as the detection result of the vibrio cholerae O1 group, and the wavelength of 550nm is shown as the detection result of the vibrio cholerae O139 group.
Example 4
Specificity detection
Test 1:
the test selects a nucleic acid sample of a common pathogen with similar infection symptoms with vibrio cholerae as a reference substance for a specificity test, and the bacterial types comprise: non-O1/O139 group Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, and Escherichia coli O157.
The virus sample and the positive quality control substance and the negative quality control substance in the kit of example 2 are used as test samples, the positive quality control substance is used as a positive control, the negative quality control substance is used as a negative control, and the test method of example 3 is adopted for detection.
The detection results are shown in fig. 1: no. 1-8 respectively correspond to detection results of non-O1/O139 groups of vibrio cholerae, vibrio parahaemolyticus, vibrio vulnificus, vibrio alginolyticus, vibrio mimicus, escherichia coli O157, positive control and negative control under the wavelengths of 520nm and 550 nm.
The experimental results show that the detection results of the reference bacteria No. 1-6 are all reported as negative, namely "-"; the test result of the negative control was reported as negative, i.e. "-"; the test result of the positive control is reported as positive, i.e. + ". The results show that the detection method provided by the invention has good specificity, and even a sample which is not the Vibrio cholerae of O1/O139 group can not be detected as false positive.
Test 2:
the test selects a nucleic acid sample of a common pathogen with similar infection symptoms with vibrio cholerae as a reference substance for a specificity test, and the bacterial types comprise: o1 group of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, and Escherichia coli O157.
The virus sample and the positive quality control substance and the negative quality control substance in the kit of example 2 are used as test samples, the positive quality control substance is used as a positive control, the negative quality control substance is used as a negative control, and the test method of example 3 is adopted for detection.
The detection results are shown in fig. 1: no. 1-8 respectively correspond to the detection results of O1 group Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Escherichia coli O157, positive control and negative control under the wavelength of 520nm and 550 nm.
The experimental result shows that the detection result of the vibrio cholerae of the group 1O 1 is positive, namely '+'; the detection results of bacteria of sample Nos. 2 to 6 were all reported as negative, i.e. "; the test result of the negative control was reported as negative, i.e. "-"; the test result of the positive control is reported as positive, i.e. + ". The results show that the detection method has good specificity and can accurately detect the O1 group vibrio cholerae.
Test 3:
the test selects a nucleic acid sample of a common pathogen with similar infection symptoms with vibrio cholerae as a reference substance for a specificity test, and the bacterial types comprise: o139 group Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, and Escherichia coli O157.
The virus sample and the positive quality control substance and the negative quality control substance in the kit of example 2 are used as test samples, the positive quality control substance is used as a positive control, the negative quality control substance is used as a negative control, and the test method of example 3 is adopted for detection.
The detection results are shown in fig. 1: no. 1-8 respectively correspond to detection results of O139 group vibrio cholerae, vibrio parahaemolyticus, vibrio vulnificus, vibrio alginolyticus, vibrio mimicus, escherichia coli O157, positive control and negative control under the wavelengths of 520nm and 550 nm.
The experimental result shows that the detection result of the O139 group vibrio cholerae No. 1 is positive, namely '+'; the detection results of bacteria of sample Nos. 2 to 6 were all reported as negative, i.e. "; the test result of the negative control was reported as negative, i.e. "-"; the test result of the positive control is reported as positive, i.e. + ". The results show that the detection method has good specificity and can accurately detect the O139 group vibrio cholerae.
Example 5
Sensitivity test
The positive control (plasmid) from example 2 was diluted in a 10-fold gradient to give a copy number of 107、106、105、104、103、102、101And 100The positive plasmid of (1) is detected by the detection method in example 3, each group is subjected to 3 times of repeated tests, and the result shows that the lowest copy number which can be effectively detected is 102The positive plasmid of (1).
Example 6
Repeatability test
The copy number of 10 in example 5 was used5And 103The positive plasmid of (1), each group was repeatedly tested 10 times, POCKITTMThe report results of the device are positive, namely "+", which shows that the iPCR detection method used by the invention has good repeatability and stability.
The embodiments of the present application have been described in detail with reference to the drawings, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
SEQUENCE LISTING
<110> Zhuhai international travel health care center (Arch North customs port outpatient department)
<120> composition, kit and detection method for detecting vibrio cholerae O1 group and O139 group
<130> 1
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence
<400> 1
cattcactta tgttgcctcg gt 22
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence
<400> 2
gactcacctt cgatttcagc a 21
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence
<400> 3
gttcccttgt tagaccaccg 20
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence
<400> 4
cctttccacc tcggtatttc a 21
<210> 5
<211> 19
<212> DNA
<213> Artificial sequence
<400> 5
aataccatag tccagtgtg 19
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
tgctgagttt gctgccagtt 20
<210> 7
<211> 200
<212> DNA
<213> Artificial sequence
<400> 7
tgtaccaaca ttcacttatg ttgcctcggt taataccata gtccagtgtg gtgcgttacc 60
cgtttttgct gaaatcgaag gtgagtctct acaagtgagc ttcattagaa gggcgggttc 120
ccttgttaga ccaccgcatt gctgagtttg ctgccagttt gccgatccat ttgaaatacc 180
gaggtggaaa gggaaagtgg 200
Claims (10)
1. A composition for detecting vibrio cholerae group O1 and O139, comprising:
the primer pair 1 for detecting the vibrio cholerae O1 group has the nucleotide sequence as follows:
a forward primer: 5'-CATTCACTTATGTTGCCTCGGT-3' the flow of the air in the air conditioner,
reverse primer: 5'-GACTCACCTTCGATTTCAGCA-3', respectively;
and a primer pair 2 for detecting vibrio cholerae O139 group, wherein the nucleotide sequence is as follows:
a forward primer: 5'-GTTCCCTTGTTAGACCACCG-3' the flow of the air in the air conditioner,
reverse primer: 5'-CCTTTCCACCTCGGTATTTCA-3' are provided.
2. The composition as claimed in claim 1, further comprising:
the probe A for detecting the vibrio cholerae O1 group has the nucleotide sequence as follows: 5'-AATACCATAGTCCAGTGTG-3', respectively;
and a probe B for detecting vibrio cholerae O139 group, wherein the nucleotide sequence of the probe B is as follows: 5'-TGCTGAGTTTGCTGCCAGTT-3' are provided.
3. The composition of claim 2, wherein the 5 'ends of probe a and probe B are labeled with a fluorescent group, and the 3' ends of probe a and probe B are labeled with a quenching group; and the fluorescent groups marked by the probe A and the probe B are different.
4. The composition of claim 3, wherein the fluorophore labeled by probe A is FAM and the fluorophore labeled by probe B is HEX.
5. A kit for detecting Vibrio cholerae group O1 and O139, comprising the composition of any one of claims 1 to 4.
6. The kit of claim 5, further comprising four nucleotides, a positive quality control substance, a negative quality control substance, a magnesium ion buffer, formamide, ammonium sulfate, trehalose, and Taq DNA polymerase, wherein the four nucleotides are dATP, dGTP, dCTP, and dUTP.
7. The kit of claim 6, wherein the positive control is a pGEM-T vector plasmid having a nucleotide sequence shown in SEQ ID No. 7.
8. A detection method for vibrio cholerae O1 group and O139 group is characterized by comprising the following steps:
extracting DNA of a sample to be detected, adding the DNA into a thermal convection PCR tube, and detecting by using the kit and the thermal convection PCR detection device of any one of claims 5 to 7.
9. The detection method according to claim 8, wherein the final concentration of each primer is 0.3-0.8 μmol/L and the final concentration of each probe is 0.2-0.4 μmol/L in the thermal convection PCR tube.
10. The detection method according to claim 8, wherein the reaction procedure of the detection method is: keeping the temperature at 50 ℃ for 8-10 min; and carrying out thermal convection PCR reaction at 95 ℃ for 45-50 min.
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