CN111996267A - Fluorescent quantitative PCR (polymerase chain reaction) primer group and kit for rapidly detecting AKK (alkyl ketene dimer) bacteria and application of fluorescent quantitative PCR primer group and kit - Google Patents
Fluorescent quantitative PCR (polymerase chain reaction) primer group and kit for rapidly detecting AKK (alkyl ketene dimer) bacteria and application of fluorescent quantitative PCR primer group and kit Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention discloses a fluorescent quantitative PCR primer group for rapidly detecting AKK bacteria and application thereof in preparing a fluorescent quantitative PCR reagent or a kit for detecting AKK bacteria, wherein the primer group comprises a forward primer and a reverse primer, and the primer sequences are shown as SEQ ID NO: 1-2; the primer group is designed according to the 16SrRNA genome sequence of AKK bacteria, and has strong specificity, high accuracy and good stability. The invention also discloses a fluorescent quantitative PCR kit for rapidly detecting AKK bacteria, and application thereof in clinical or scientific research detection of AKK bacteria, wherein the kit comprises the specific primer group, optimizes a reaction system and reaction conditions, can specifically and quantitatively detect AKK bacteria from a complex sample, and has no cross reaction with other bacteria; the accuracy is high, the repeatability is good, the sensitivity is strong, and the kit can be used for quickly, sensitively and accurately quantitatively detecting the AKK bacteria in the intestinal flora.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a fluorescent quantitative PCR primer group for rapidly detecting AKK (alkyl ketene dimer) bacteria, a kit and application thereof.
Background
The intestinal flora is a huge number of microbial populations in human intestinal tracts, and the composition and change of the intestinal flora are closely related to the health condition of a host, so that certain scientific basis can be provided for preventing and treating related diseases by observing the change of the intestinal flora. At present, the detection method of the abundance of the intestinal flora mainly comprises two major methods, namely a gene sequencing method and a molecular biological method. The gene sequencing method is a common method for researching the abundance of microorganisms, but has the problems of long time consumption, high cost, multiple influence factors and the like; the molecular biology technology method has the advantages of short detection time, high sensitivity, accurate result and the like, makes up for many defects of a sequencing method, and is widely applied to the research of microbial detection. Among molecular biological detection methods, the fluorescence quantitative PCR technology has the characteristics of rapidness, quantifiability, high sensitivity, good repeatability, capability of detecting dead and live bacteria and the like, and becomes one of important molecular biological methods in intestinal microecological research. Although there are many reports on establishment and application of quantitative PCR detection methods at home and abroad, the research on the quantitative PCR detection method of AKK bacteria in excrement is not available at present.
AKK bacteria are the original bacteria in human intestinal tract, but because of its low content in intestinal tract and inability to culture in aerobic environment, it was not identified by scientists until 2004. Compared with bifidobacterium and lactobacillus, AKK is absolutely a primary thastream of cottage but over-potent trichome. In the research of various diseases of human bodies, AKK bacteria is found to be a beneficial bacterium: the level of AKK bacteria in obese people is significantly lower than that of healthy people, and the abundance of AKK bacteria has positive correlation with insulin sensitivity and healthier metabolic state; the immunotherapy of cancer patients with high AKK bacteria content in intestinal tract has better treatment effect. By adopting a strict random, double-blind and placebo-controlled human experiment, the Cani team of the university of Luwenun, Belgium discovers that the AKK bacteria can be orally taken to reduce the weight, improve the insulin sensitivity, reduce the insulinemia and the like.
Therefore, there is an urgent need in the art to develop a rapid, specific and quantitative AKK bacteria detection means, which provides technical support for AKK bacteria screening and research related to intestinal flora.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a fluorescent quantitative PCR primer group for rapidly detecting AKK bacteria, which is designed according to the 16SrRNA genome sequence of the AKK bacteria, and has strong specificity, high accuracy of AKK bacteria amplification and good stability;
the second purpose of the invention is to provide an application of the primer group in the preparation of a fluorescent quantitative PCR reagent or a kit for detecting AKK bacteria.
The invention also aims to provide a fluorescent quantitative PCR kit for rapidly detecting AKK bacteria, which comprises the specific primer group, optimizes a reaction system, can specifically detect the AKK bacteria from a sample, and has no cross reaction with other bacteria except the AKK bacteria; the accuracy is high, the repeatability is good, the sensitivity is strong, and the kit can be used for quickly, sensitively and accurately quantitatively detecting the AKK bacteria in the intestinal flora.
The fourth purpose of the invention is to provide the application of the kit in clinical or scientific research detection of AKK bacteria, further optimize PCR amplification conditions, have good log-linear relationship between the strength of a fluorescence signal and a template amplification product, and have high quantitative detection accuracy; and the method is simple to operate, high in automation degree, less in cross contamination, and clear and visual in result interpretation.
One of the purposes of the invention is realized by adopting the following technical scheme:
a fluorescent quantitative PCR primer group for rapidly detecting AKK bacteria comprises a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown as SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO: 2, respectively.
The second purpose of the invention is realized by adopting the following technical scheme:
the application of the fluorescent quantitative PCR primer group for rapidly detecting AKK bacteria in the preparation of a fluorescent quantitative PCR reagent or a kit for detecting AKK bacteria.
The third purpose of the invention is realized by adopting the following technical scheme:
a fluorescence quantitative PCR kit for rapidly detecting AKK bacteria comprises the primer group; wherein the volume ratio of the forward primer to the reverse primer is 1: 1.
Further, the kit also comprises SYBR Green Reaction Mix, ROX and sterile water.
The fourth purpose of the invention is realized by adopting the following technical scheme:
the application of the fluorescent quantitative PCR kit for rapidly detecting AKK bacteria in clinical or scientific research detection of AKK bacteria comprises the following steps:
s1: extracting the genome DNA of the sample;
s2: putting the genome DNA into a PCR reaction tube, adding a primer group, SYBR Premix Ex Taq, ROX and sterile water into the tube, and carrying out PCR amplification and melting curve determination.
Further, the reaction conditions of the PCR amplification are as follows: 5min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 30s, 40 cycles.
The conditions for measuring the melting curve are as follows: cooling to 55 deg.C for 1min at 95 deg.C, and heating to 95 deg.C; the fluorescence signal was collected throughout the procedure.
After the fluorescent quantitative PCR amplification, all samples with amplification curves above the threshold are positive samples, and the abundance of AKK bacteria in the samples can be calculated according to the Ct value of the amplification curves and the standard curve, so that the result is simple and visual to interpret.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the 16SrRNA gene sequence of the AKK bacteria, the specific primers are designed, so that the AKK bacteria can be specifically and quantitatively detected from excrement; the reaction system and the amplification condition are optimized, the repeatability for detecting the AKK bacteria is good, and the method is a favorable means for quickly, sensitively and accurately detecting the abundance value of the AKK bacteria in intestinal flora.
2. The reaction system and the amplification condition of the invention are reasonably designed, the strength of the PCR fluorescent signal has good logarithmic linear relation with the template amplification product, the initial template concentration of the sample can be quantified through the detection of the PCR fluorescent signal, the error is small, the trace change of the abundance of a small amount of AKK bacteria in the intestinal flora can be detected, and the quantitative sensitivity is high.
3. The kit disclosed by the invention is simple to operate and high in automation degree when being applied, amplification and detection of a PCR product are completed in one step under the condition of closed tube, a cover does not need to be opened, the chances of cross contamination and environmental pollution are less, and the probability of result deviation is reduced; the detection result is also clear and intuitive to interpret. Meanwhile, the whole system does not contain toxic and harmful substances, and is harmless to operators and the environment.
Drawings
FIG. 1 is a photograph of agarose gel electrophoresis of the PCR amplification product of example 1;
FIG. 2 is a standard curve of fluorescence quantitative PCR of AKK bacteria of the present invention;
FIG. 3 shows the melting curve of the fluorescence quantitative PCR in example 2.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
A fluorescent quantitative PCR primer group for rapidly detecting AKK bacteria comprises a forward primer and a reverse primer; wherein, the nucleotide sequence (shown as SEQ ID NO: 1) of the forward primer AKK-F is:
5’-CAGCACGTGAAGGTGGGGAC-3’
the nucleotide sequence of the reverse primer AKK-R (shown as SEQ ID NO: 2) is as follows:
5’-CCTTGCGGTTGGCTTCAGAT-3’
the Primer is designed according to the 16SrRNA gene sequence of the AKK bacteria, and the Primer can only be combined with the AKK bacteria and has strong specificity as shown by an NCBI Primer-Blast comparison result.
The fluorescent quantitative PCR primer group for rapidly detecting AKK bacteria can be applied to preparation of a fluorescent quantitative PCR reagent or a kit for detecting AKK bacteria.
A fluorescence quantitative PCR kit for rapidly detecting AKK bacteria comprises the primer group; wherein the volume ratio of the forward primer to the reverse primer is 1: 1.
The kit also comprises SYBR Green Reaction Mix, ROX and sterile water.
The application of the fluorescent quantitative PCR kit for rapidly detecting AKK bacteria in clinical or scientific research detection of AKK bacteria comprises the following steps:
s1: extracting the genome DNA of the sample;
s2: putting the genome DNA into a PCR reaction tube, adding a primer group, SYBR Premix Ex Taq, ROX and sterile water into the tube, and performing PCR amplification and melting curve determination; and reading the Ct value of the amplification, and calculating the abundance value of the AKK bacteria according to the standard curve.
Wherein, the reaction conditions of the PCR amplification are as follows: 5min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 30s, 40 cycles. The conditions for melting curve measurement were: cooling to 55 deg.C for 1min at 95 deg.C, and heating to 95 deg.C; the fluorescence signal was collected throughout the procedure. After the fluorescent quantitative PCR amplification, the sample with the amplification curve above the threshold value is a positive sample, and the abundance of the AKK bacteria in the sample can be calculated according to the Ct value of the amplification curve and the standard curve, so that the result is simple and visual to interpret.
The invention utilizes the designed primer group to carry out common PCR amplification on the genome DNA of the standard strain of the bacteria to be detected, optimizes reaction parameters, makes an amplification product be a single and clear strip, and obtains the minimum Ct under the condition of ensuring that the melting curve of a sample is a standard single peak.
Example 1
Primer specificity analysis
1) Sample processing
Human feces were used as a sample, and a Bacterial genome extraction kit (QIAgene Bacterial genome extraction kit) was used to extract human feces genomic DNA as a template to be amplified.
2) PCR amplification
The reaction system of this example comprises: 2 XPCR Master Mix10.0 μ L, AKK-F0.8 μ L, AKK-R0.8 μ L, 2.0 μ L template to be amplified, sterile water to make up to 20 μ L.
AKK-F：5’-CAGCACGTGAAGGTGGGGAC-3’；
AKK-R：5’-CCTTGCGGTTGGCTTCAGAT-3’。
Carrying out PCR amplification according to the reaction system under the following reaction conditions: 5min at 95 ℃; 95 ℃ for 15sec, 60 ℃ for 30sec, 40 cycles.
3) Product validation
Performing agarose gel electrophoresis on the product obtained by amplification in the step 2), wherein the result is shown in figure 1;
and (3) performing gel recovery and sequencing on the PCR product, wherein the sequence obtained by sequencing is as follows: CAGATCAACTGGGAGGAAGGTGGGGACGACGTCAGGTCAGTATGGCCCTTATGCCCAGGGCTGCACACGTACTACAATGCCCAGTACAGAGGGGGCCGAAGCCGCGAGGCGGAGGAAATCCTAAAAACTGGGCCCAGTTCGGACTGTAGGCTGCAACCCGCCTACACGAAGCCGGAATCGCTAGTAATGGCGCATCAGCTACGGCGCCGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACATCATGGAAGCCGGTCGCACCCGAAGTATCTGAAGC
As can be seen from FIG. 1, the gel electrophoresis shows that the PCR product is a single band, which is clear, and no other bands are present. The sequencing result of the amplified product is a single peak and has no impurity peak, and the PCR product sequence is AKK bacteria through NCBI blast comparison. The primer of the invention has specificity, and the reaction system has no cross reaction with other bacteria except the bacteria to be detected.
Example 2
Establishment of a Standard Curve
1) AKK bacterial liquid preparation and gDNA extraction
Operating in an anaerobic workstation, recovering the AKK bacteria standard strain by using a BHI culture medium, inoculating 5 percent of the strain, and placing the strain in an anaerobic incubator for culturing for 48 hours. The objective AKK genomic DNA was extracted using a Bacterial genome extraction kit (QIAgene Bacterial genome extraction kit).
2) Drawing of standard curve
Performing 10-fold gradient dilution on the AKK genomic DNA obtained in the step 1), and performing fluorescent quantitative PCR amplification on a qPCR instrument of Thermofish QuantStudio by taking the AKK genomic DNA with different gradient concentrations as templates to be amplified.
The 20. mu.L reaction system of this example comprises: SYBR Green Reaction Mix10. mu.L, Rox 0.4. mu.L, AKK-F0.8. mu. L, AKK-R0.8. mu.L, AKK genomic DNA template 1. mu.L, sterile water 7.0. mu.L;
AKK-F：5’-CAGCACGTGAAGGTGGGGAC-3’；
AKK-R：5’-CCTTGCGGTTGGCTTCAGAT-3’。
the PCR conditions in this example were: pre-denaturation at 95 ℃ for 5 min; 95 ℃ for 15sec, 60 ℃ for 30sec, for 40 cycles.
The melting curve analysis conditions in this example were: cooling to 55 ℃ at 95 ℃ for 1min, then heating to 95 ℃, and collecting fluorescence signals in the whole process.
And establishing a standard curve according to the Ct values of the obtained AKK genome DNA with different concentrations and obtaining a regression equation in a corresponding linear range. As can be seen from the results of PCR detection, the amount of the DNA initiation template of the AKK standard strain was 102～105When the molecular weight is within the range of copies/mu L, a good linear relation exists between the template amount and the Ct value, all correlation coefficients are greater than 0.99, and as shown in figure 2, the quantitative detection result is accurate. Meanwhile, the lowest quantitative detection concentration of AKK bacteria is 102copies/μL。
The melting curve of the amplification product is shown in FIG. 3, and 10 is shown in FIG. 32～105The melting curves of the amplification products in the range of copies/mu.L are all unimodal and have no hetero-peak, which indicates that the melting curve is 102～105Primers are more specific in the range of copies/. mu.L.
Example 3
Detection of AKK bacteria in fecal samples
1) Sample processing
Randomly selecting 2 volunteers, collecting feces as samples for three weeks, and extracting total genomic DNA of each sample with a Bacterial genome extraction kit (QIAgene Bacterial genome extraction kit).
2) Fluorescent quantitative PCR amplification
This example 20. mu.L of the reaction system included: 2 × SYBR Green Reaction Mix10 μ L, Rox Reference Dye 0.4 μ L, AKK-F0.8 μ L, AKK-R0.8 μ L, AKK genomic DNA template 1 μ L, sterile water 7.0 μ L;
AKK-F：5’-CAGCACGTGAAGGTGGGGAC-3’；
AKK-R：5’-CCTTGCGGTTGGCTTCAGAT-3’。
the PCR conditions in this example were: pre-denaturation at 95 ℃ for 5 min; 40cycles of 95 ℃ for 15sec and 60 ℃ for 30 s;
the melting curve analysis conditions in this example were: cooling to 55 ℃ at 95 ℃ for 1min, then heating to 95 ℃, and collecting fluorescence signals in the whole process.
Performing fluorescent quantitative PCR amplification on a qPCR instrument of Thermofish QuantStudio by using the total genomic DNA of the volunteer stool sample obtained in the step 1) as a template by adopting the reaction system and the PCR reaction condition.
The detection results show that AKK bacteria in all the fecal samples are positive, corresponding Ct values are read, and the abundance (cfu/g) change of the AKK bacteria contained in the feces of the volunteers at different times is calculated by using the standard curve established in the embodiment 2, and is shown in the table 1. As can be seen from Table 1, the Ct values detected by the same volunteer within three weeks have no significant difference, and the AKK bacteria detection stability of the invention in practical samples is good. Furthermore, the melting curves of the total genomic DNA of the fecal samples of the two volunteers after PCR amplification are both single peaks and have no impurity peaks, which indicates that no non-specific amplification occurs in the embodiment, and the specificity of the primer of the invention is better.
TABLE 1 Ct values for qPCR amplification and abundance tables for AKK bacteria
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the scope of the present invention claimed in the present invention.
SEQUENCE LISTING
<110> Kangmeihua Dagenetechnology Co., Ltd
<120> fluorescent quantitative PCR primer group and kit for rapidly detecting AKK bacteria and application thereof
<130> 20200715
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 2
Claims (7)
1. A fluorescent quantitative PCR primer group for rapidly detecting AKK bacteria is characterized by comprising a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown as SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO: 2, respectively.
2. The primer group of claim 1 is applied to the preparation of a fluorescent quantitative PCR reagent or a kit for detecting AKK bacteria.
3. A fluorescent quantitative PCR kit for rapidly detecting AKK bacteria, which is characterized by comprising the primer group of claim 1; wherein the volume ratio of the forward primer to the reverse primer is 1: 1.
4. The kit of claim 3, further comprising a SYBR Green Reaction Mix, ROX and sterile water.
5. The use of the kit of claim 4 for clinical or scientific detection of AKK bacteria, comprising the steps of:
s1: extracting the genome DNA of the sample;
s2: putting the genome DNA into a PCR reaction tube, adding a primer group, SYBR Premix Ex Taq, ROX and sterile water into the tube, and carrying out PCR amplification and melting curve determination.
6. The use according to claim 5, wherein the PCR amplification is performed under the following conditions: 5min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 30s, 40 cycles.
7. Use according to claim 5, wherein the conditions for melting curve determination are: cooling to 55 deg.C for 1min at 95 deg.C, and heating to 95 deg.C; the fluorescence signal was collected throughout the procedure.
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| CN115747357A (en) * | 2022-11-15 | 2023-03-07 | 迪辅乐生物(上海)有限公司 | Detection kit and detection method for intestinal mucinous-Ackermanella |
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