CN112458193A - Intestinal flora nucleic acid detection kit based on PCR-quantum dot fluorescence method and detection method - Google Patents
Intestinal flora nucleic acid detection kit based on PCR-quantum dot fluorescence method and detection method Download PDFInfo
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Abstract
The invention provides a kit and a method for detecting nucleic acid of intestinal flora based on a PCR-quantum dot fluorescence method, and provides corresponding primers and probes, so that detection of 32 intestinal flora can be comprehensively and rapidly performed, the existence of the 32 intestinal flora can be obtained simultaneously through one-time detection for the first time, and the 32 intestinal flora contains common probiotics and pathogenic bacteria corresponding to different diseases.
Description
Technical Field
The invention belongs to the field of biological and new medical technology biochips, relates to a third product in an in vitro diagnostic reagent, and particularly relates to an intestinal flora nucleic acid detection kit and a detection method based on a PCR-quantum dot fluorescence method, which can simultaneously detect 35 human intestinal common flora.
Background
The intestinal tract, called the second "brain" of the human body, is the largest organ of the human body that expels toxins, and is responsible for most of the expelling of toxins and for the resistance to diseases. Even when a person is at rest, the intestine is still in motion and, in addition to digesting and absorbing food, it is also involved in the regulation of many important organ functions. Thus, intestinal health is gaining more and more attention. In recent years, with the development of the scientific and technical level, especially the development of the molecular biotechnology level, a great deal of research shows that the intestinal flora plays an extremely important role in the health of a host, so that the importance of the intestinal flora is generally accepted.
1.1 how does the gut flora influence host health and how does it interact with the host? The details are as follows:
1.1.1 composition, Structure and distribution of intestinal flora
The human intestinal tract colonizes 1010 different microorganisms, including bacteria, fungi, viruses and protozoa, 10 times as many as all cells of the human body, at least 150 times as many as the total number of genes in the human genome, and the genomic information they contain is called the "second genome" of the human. The human intestinal microorganisms are mainly bacteria, and have 450 types on average, so that a huge and complex ecosystem can be formed, and the system and the human body have an inseparable mutual and beneficial symbiotic relationship. With the continuous deepening of molecular biology technical means, intestinal flora symbiotic in human bodies is found at present to mainly comprise four major groups of bacteroidetes, firmicutes, proteobacteria and actinomycetes, and most of the bacteria belong to obligate anaerobes. Wherein, the bacteroidetes and the firmicutes account for higher proportion and account for more than 90 percent of the intestinal bacteria.
1.1.2 physiological Functions of the intestinal flora
The intestinal flora, called the human "second brain", is closely related to human health. These microorganisms grow in the human gut and form an extremely complex micro-ecosystem, the diversity of gut microorganisms may be the result of a strong selection and co-evolution between the host and gut microorganisms. The intestinal microorganisms are closely related to hosts in genomics, immunology and metabonomics, and play a great role in the health and disease development of human bodies.
(1) Nutrition and substance metabolism
The normal flora colonized in the intestinal tract can provide the host with necessary nutrient elements for synthesizing B, K vitamins, biotin, folic acid and the like. The intestinal flora contains abundant genes which are generated by the long-term evolution of human beings and are peculiar to microorganisms, such as genes participating in the metabolism of saccharides, amino acids, vitamins, cholesterol and exogenous compounds, genes generated by methane and the like, and the genes supply various metabolic enzymes to biochemical metabolic pathways of an organism so as to promote the metabolism of a human body.
(2) Biological barrier function
The intestinal mucosa barrier is the first defense line for the body to defend against exogenous invasion, and the normal flora of the intestinal mucosa epithelium is an important component of the colon mucosa biological barrier, so that the complex interaction of infected intestinal microorganisms caused by the colonization of potential pathogenic bacteria or external-attacking bacteria on the mucosa, the intestinal physiological barrier and a host immune system can be inhibited, and great contribution is made to the stabilization of the intestinal environment.
(3) Regulating and controlling body's immune response function
Under normal conditions, the intestinal mucosa immune system has the function of defending pathogenic factors and pathogenic flora from invading the intestines. If the intestinal immune system is damaged, the host can lack the tolerance to intestinal symbiotic bacteria, so that antigenic substances such as intestinal contents stimulate intestinal mucosa, the intestinal immune system is further stimulated, abnormal immune response is generated, and finally some diseases are induced. As an important component of the intestinal mucosa, the normal flora of the intestinal tract stimulates a host immune system through bacteria and metabolites thereof to activate immune cells, and improves the immunity of the organism by generating antibodies, regulating phagocytosis, increasing the generation of interferon and the like.
(4) Participate in regulating central nervous system activity
In recent years, the role of the gut flora on brain function has become more and more interesting. Research shows that the influence of intestinal flora on a host is only shown in gastrointestinal tracts, and the intestinal flora-intestinal brain axis can be formed through 3 paths (neuroendocrine, vagus nerve and immune paths) of the intestinal brain axis, so that the intestinal flora-intestinal brain axis can have great influence on brain functions and behaviors and participate in the regulation and control of central nervous system activities such as stress response, brain development, depression, anxiety, cognitive function and the like.
(5) Regulating intestinal peristalsis
The intestinal flora has a regulating effect on gastrointestinal tract movement, is directly or indirectly involved in energy metabolism of 3 nutrient substances, is also involved in vitamin synthesis, and is also influenced in absorption and distribution of fat. The bifidobacteria in the intestinal tract can promote the intestinal tract movement by adjusting the pH value of the intestinal tract, thereby reducing the contact time of pathogenic bacteria and intestinal tract adhesion and promoting the drainage of the pathogenic bacteria.
1.1.3 intestinal flora and disease
Microorganisms colonized in animal intestinal tracts can maintain the health of hosts through various physiological metabolic pathways and play various physiological functions, and if the structure and the quantity of intestinal flora are changed, the homeostasis of the intestinal tract can be influenced, so that the function of the intestinal flora is abnormal, and further various diseases such as diabetes, obesity, intestinal stress syndrome, inflammatory bowel disease, liver disease, central nervous system disease, cancer and the like are caused, so that the interaction between the intestinal flora and the hosts is influenced to a certain extent. The relationship of the intestinal flora to diabetes and obesity is mainly explained below.
(1) Intestinal flora and diabetes
In recent studies on obesity and type 2 diabetes caused by imbalance of intestinal flora, it is considered that reduction of probiotics such as intestinal lactic acid bacteria and bifidobacteria is closely related to impaired glucose tolerance in many views. The imbalance of intestinal flora can induce the abnormal level and composition of SCFAs in the body, so that the anti-inflammatory response capability of the intestinal tract, the secretion of ghrelin hormone and other factors are affected, and the function of islet cells and the insulin resistance are damaged. The intestinal bacterial flora structure of type 2 diabetics is different from that of normal people. The 2012's major gene and Shenzhen's second hospital and other units are combined to complete the metagenome association analysis of the intestinal microorganisms and the type 2 diabetes, and the moderate intestinal microecological disorder phenomenon and the deficiency of butyric acid-producing bacteria in the body of the type 2 diabetes patient in China are found. Meanwhile, the antagonistic relationship exists between beneficial flora and harmful flora, which is particularly obvious between different flora of clostridium.
(2) Intestinal flora and obesity
Over the last decade, more and more studies have shown that the intestinal flora is closely related to the development of obesity. Fei et al demonstrated by Koch's Law that Enterobacter cloacae, an opportunistic pathogen in the gut from a severely obese patient, Enterobacteriaceae, is able to cause obesity and insulin resistance in sterile mice, directly explaining the causal relationship between gut flora and obesity from the level of the strain. Studies have shown that changes in the early gut microbiome of newborns are closely related to obesity, autoimmune functions including asthma, allergy, and even type 1 diabetes in their childhood. Numerous studies have shown that subtle specific changes in the intestinal flora of overweight or obese persons will cause changes in obesity, inflammation, glucose and lipid balance, both positive and negative.
1.1.4 factors affecting the structure of intestinal flora
In the process of interaction between the intestinal flora and the host, the formation and the diversity composition of the intestinal flora are influenced by the conditions of the host and environmental factors such as regions, dietary habits and the like. At present, the research finds that the main factors influencing the structure of the intestinal flora are as follows: host self-gene, age and weight; national, regional, cultural factors; sex, genetic background factors; dietary habit factors; other pharmaceutical factors such as antibiotics; surgery and other external force factors.
1.2 research progress of intestinal flora detection technology at home and abroad
With the rapid development of science and technology, people learn and research the intestinal flora gradually, and the 'second human genome' of the intestinal flora gradually uncovers the face yarn. The intestinal flora is of many species and is remarkable in quantity and rich in function. The detection of the intestinal flora is mainly to determine the amount and the type of the intestinal flora. The common methods include the most basic and traditional bacterial culture methods, and the polymerase chain reaction technology and the 16S rDNA fingerprint spectrum technology based on PCR are widely used at present. And some emerging technologies such as fluorescence in situ hybridization, gene chips, metagenome sequencing technology and the like.
1.2.1 bacterial culture
Bacterial culture is the most traditional bacterial study method, and gastrointestinal bacteria are cultured using various selection media, and the number of colonies is determined by dilution by multiple ratio and colony counting. As a basic method for biological identification, the traditional culture method plays an important role in detecting intestinal flora, but has the disadvantages that for intestinal micro-ecosystems with various and large quantities, analysis on partial flora is not comprehensive enough, the relation between the whole micro-ecosystem and disease occurrence and development cannot be reflected, and the analysis result and conclusion have certain limitations, so the bacterial culture method is not suitable for intestinal flora analysis. However, the recent research finds that the mixed culture technology is expected to break through the problems of the prior bacteria isolated culture. At present, the transition from pure culture to mixed culture relies mainly on the progress of three technologies: the micro-fluid technology, the next generation 3D biological printing and the single cell metabonomics are expected to realize the systematic large-scale symbiotic culture research of three or more microorganisms by the progress of the technologies in the future.
1.2.216 s rRNA polymerase chain reaction technique (16s rRNA PCR)
The 16S rRNA gene is closely related to most microorganisms, has strong variability and high conservation, and has high conservation, so that the 16S rRNA gene can be amplified by using the characteristic to design a universal primer of the bacteria so as to obtain 16S rDNA fragments, and the fragments are sequenced and subjected to germ line analysis and then compared with data in a 16S rRNA database so as to identify the microorganism types in a sample. The advantage of this technique is that bacteria in the intestine which are not able to culture or grow slowly can be detected. The disadvantage is that cross-amplification reaction is easily caused in the PCR amplification process, so that false positive result is generated. Therefore, the operator is required to strictly follow the operating rules, continuously accumulate the experience of observing and judging the result, eliminate the interference and ensure the accuracy of the detection result. 1.2.3 PCR-based 16S rDNA fingerprinting technology
The 16S rDNA fingerprint spectrum technology based on the PCR mainly comprises a Denaturing Gradient Gel Electrophoresis (DGGE) technology, a Temperature Gradient Gel Electrophoresis (TGGE) technology and the like. The basic process of denaturing gradient gel electrophoresis for detecting intestinal flora includes extracting DNA from intestinal tract sample and amplifying 16SrDNA with PCR process as common primer. The amplification product contained the 16SrDNA fragment of all enteric bacteria present in this sample. When 16SrDNA fragments of different bacteria pass through DGGE gel, partial denaturation can occur in polyacrylamide gel with different urea and formamide concentration gradients, and the migration speed is obviously reduced, so that the 16SrDNA fragments can be separated. Isolation of the 16SrDNA fragment resulted in a map of the bacterial population, each DNA fragment type representing a species. The technology has the advantages that the method can detect the flora with less gastrointestinal tract flora content; disadvantages are, for example, that DGGE requires DNA length (generally below 500 bp) and usually only shows the dominant flora, which does not reflect the abundance of the microorganisms, and that the technical equipment and experimental materials are expensive and the formamide gradient gel used is toxic.
The basic principle of TGGE is that after PCR of 16SrRNA gene, the amplified product has GC fragments with higher melting temperature, and when electrophoresis is carried out in gels with different temperature gradients, due to different melting speeds and degrees of DNA molecules with different sequences, the DNA molecules stop migrating at different positions of the gel, so that the DNA molecules with different sequences are separated and fingerprint print is left. The application of the technology can rapidly detect the constitution of the flora of the separate flora. However, as with DGGE, the use of toxic gels in the experiments is a disadvantage of this technique.
1.2.4 Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR)
A fluorescence reporter group and a quenching group are added when a primer is designed aiming at the 16SrRNA gene of the intestinal bacteria, 1 fluorescence signal is correspondingly generated when every 1 specific PCR product appears, and how many amplification products exist can be detected by an instrument receiving the fluorescence signals. The method has the advantages of high sensitivity, rapidness and quantification, and in addition, the automatic operation improves the working efficiency, the reaction is rapid, the repeatability is good, and the result is clear. The disadvantage of this technique is that the size of the amplified product cannot be monitored because of the use of a closed assay, which reduces the number of detection steps for electrophoresis after amplification. Because of the kinds of fluorescein and the limitation of the detection light source, the QPCR experiment is relatively expensive, thereby relatively limiting its wide application.
1.2.5 fluorescence in situ hybridization technique
The Fluorescence In Situ Hybridization (FISH) technology is a technology for detecting the existence and abundance of a microorganism population by hybridizing a fluorescence-labeled specific oligonucleotide fragment serving as a probe with a DNA molecule in a target genome according to population-specific DNA sequences of known microorganisms on different classification levels. The FISH can provide the information of the morphology, the quantity and the spatial distribution of the microorganisms, and has the advantages of safety, rapidness, high sensitivity, simple and visual detection result and the like. However, the fluorescence in situ hybridization technique is limited by that the technique can only detect known strains, but cannot detect unknown strains, and most of the intestinal flora belongs to unknown strains; in addition, for bacteria in the resting stage or when the bacteria are spores, actinomycetes, the membrane permeability of the bacteria or cells is reduced, which affects the abundance estimation of the bacteria.
1.2.6 high throughput sequencing technology
High-throughput sequencing is also known as next-generation sequencing, can meet the requirement of sequencing hundreds of thousands to millions of DNA molecules at a time, and is widely applied to the research of intestinal microorganisms due to the characteristics of digital signals, high data throughput, high sequencing depth, high accuracy and the like, particularly, 16S rRNA sequencing can identify the microorganisms from the level of the strains and detect new strains, thereby greatly promoting the exploration process of people on unknown microorganisms. The development of the technology enables researchers to study and understand the microbial world from a wider and deeper angle, not only can realize synchronous sequencing of multiple variable regions of multiple samples, but also improves the sequencing speed and enlarges the sequencing flux.
1.2.7 Gene chip technology
The gene chip is also called DNA microarray, which is a new molecular biology technology developed on the basis of molecular hybridization technology. The intestinal bacteria 16S ANA gene is used as a target gene for detection, oligonucleotide probes aiming at different genera are designed to prepare a gene chip, and the human intestinal flora can be detected through hybridization reaction. The method is an analysis method capable of detecting various bacteria or different samples simultaneously, has the characteristics of high throughput, automation, rapid detection and the like, and can draw a conclusion by direct observation for simple detection or scientific experiments due to the small number of genes to be analyzed.
At present, a simple, quick, economic and high-throughput detection technology is urgently needed clinically, and a detection kit combining a gene chip with a quantum dot fluorescence method is developed on the basis of an optimized multiplex PCR system to meet the requirement. The gene chip has the characteristics of high detection flux and good specificity, can detect the technical advantages of a plurality of targets and a plurality of sites at one time, combines the advantages of large fluorescent particles of quantum dots, strong coupling capacity and high fluorescence intensity, and can detect by a light source with common wavelength, combines two detection technologies with respective advantages together to form a unique PCR-quantum dot fluorescence method for detecting nucleic acid by the high-flux gene chip, and initiates a new detection method.
The kit provided by the invention can detect 32 intestinal flora, realizes the purpose of high-throughput detection, and can simultaneously detect multiple probiotics and pathogenic bacteria in human intestinal tracts in one sample. The 32 bacterial populations are shown in table 1 below.
Table 132 intestinal flora list
| Bacterial species name | For short | Bacterial species name | For short |
| Lactobacillus bulgaricus | Lbu | Genus verrucomicrobia | Ver |
| Lactobacillus reuteri | Lre | Clostridium difficile | Cdi |
| Lactobacillus rhamnosus | Lrh | Clostridium perfringens | Cpe |
| Lactobacillus acidophilus | Lac | Genus coprinus | Cop |
| Bifidobacterium pseudocatenulatum | Pse | Genus dorsalomyces | Dor |
| Bifidobacterium longum | Lon | Genus enterococcus | EF |
| Bifidobacterium adolescentis | Ado | Enterobacter sakazakii | Esa |
| Multirow bacteroids | Bth | Genus Fusobacterium | Fus |
| Bacteroides ovorans | Bov | Proteobacteria | Pro |
| Clostridium butyricum | But | Salmonella | Sal |
| Clostridium pralatanorum | Fae | Shigella | Shi |
| Genus Ackermansia | Akk | Genus veillonella | Vei |
| Eubacterium genus | Eub | Candida albicans | CA |
| Prevotella vulgaris | Pre | Candida tropicalis | CT |
| Genus Ruminococcus | Rum | Candida krusei | CK |
| Genus Satetra | Sut | Candida glabrata | CG |
Disclosure of Invention
The invention aims to provide an intestinal flora nucleic acid detection kit and a detection method based on a PCR-quantum dot fluorescence method aiming at the defects of the prior art, and the kit and the detection method can be used for efficiently, quickly and accurately detecting 32 intestinal flora.
The technical scheme adopted by the invention is as follows:
a set of primers and a probe for detecting nucleic acid of intestinal flora comprises amplification primer sequences suitable for 32 intestinal flora detections:
16SF 1: CCGGAGGAAGGTGGG (namely SEQ ID No.1)
16SR 1: TGATCCGCGATTACTAG (namely SEQ ID No.2)
Or
16SF 2: GGTGGGGATGACGTCA (namely SEQ ID No.3)
16SR 2: CGATTACTAGCGATTCC (namely SEQ ID No.4)
And
VVC-F1: TTGAACGCACATTGCGCC (namely SEQ ID No.5)
VVC-R1: TCCTACCTGATTTGAGGTC (i.e. SEQ ID No.6)
Or
VVC-F2: CATGCCTGTTTGAGCGTC (namely SEQ ID No.7)
VVC-R2: TTGATATGCTTAAGTTCAG (namely SEQ ID No.8)
And probe sequence from 5 'end to 3' end:
lbu: GCGAGGGTAAGCGGATCTC (namely SEQ ID No.9)
And Lre: CGAGAGTAAGCTAATCTCTTA (namely SEQ ID No.10)
Lrh: CAACGAGTTGCGAGACCG (namely SEQ ID No.11)
Lac: ACAATGGACAGTACAACGAG (namely SEQ ID No.12)
Pse: ACAATGGCCGGTACAAAGAG (namely SEQ ID No.13)
Lon: GGCCGGTACAAAGAGAAGC (namely SEQ ID No.14)
Ado: GCGGATCCCTTAAAACCG (namely SEQ ID No.15)
Bth: TACCTGGTGACAGGATGCTA (namely SEQ ID No.16)
And (4) Bov: ATCCCAAAAACCTCTCTCAG (namely SEQ ID No.17)
But: CCTCGCGAGAGTGAGCAAAA (namely SEQ ID No.18)
Fae: CAAAACTCAGAAACAACGTCC (namely SEQ ID No.19)
Akk: CAGAGGGGGCCGAAGCCGCGA (namely SEQ ID No.20)
Eub: CAAAGAGAAGCGAAACCGCGA (namely SEQ ID No.21)
Pre: AAAGTCGGATGCCCGTAAG (namely SEQ ID No.22)
Rum: TGTTAACAGAGGGAAGCAAAA (namely SEQ ID No.23)
Sut: CCCAGAAAACCGATCGTAGT (namely SEQ ID No.24)
Ver: GGAAATCCTCAAAACTGGGCT (namely SEQ ID No.25)
Cdi: GTAGTACAGAGGGTTGCCAAG (namely SEQ ID No.26)
Cpe: TGGTACAGAGAGATGCAATAC (namely SEQ ID No.27)
Cop: GAAGCGAAGCGCGAGGTGGA (namely SEQ ID No.28)
Dor: CTCGCGAGGGTAAGCAAATCT (namely SEQ ID No.29)
EF: GAAACCGCGAGGTGGAGCGAAT (namely SEQ ID No.30)
Esa: TGCGTCGTAGTCCGGATTGGAG (namely SEQ ID No.31)
Fus: AGTCGCAAAGCTGTGAAGTGG (namely SEQ ID No.32)
Pro: ACCTCGCGAGAGCAAGCGGAA (namely SEQ ID No.33)
Sal: CGACCTCGCGAGAGCAAGCG (namely SEQ ID No.34)
Shi: GCGTCGTAGTCCGGAGGGGA (namely SEQ ID No.35)
Vei: CAAACCCCAGAAACAAGCTC (namely SEQ ID No.36)
CA: GCAATACGACTTGGGTTTGC (namely SEQ ID No.37)
CT: GAATTTAACGTGGAAACTTAT (namely SEQ ID No.38)
CK: CGACGTGTAAAGAGCGTCGG (namely SEQ ID No.39)
CG: CAGTATGTGGGACACGAGCG (namely SEQ ID No.40)
The corresponding 32 intestinal flora are in turn: lactobacillus bulgaricus, lactobacillus reuteri, lactobacillus rhamnosus, lactobacillus acidophilus, bifidobacterium pseudocatenulatum, bifidobacterium longum, bifidobacterium adolescentis, bacteroides multiradiae, bacteroides ovatus, clostridium butyricum, clostridium pratensis, eckmann species, eubacterium species, prevotella, ruminococcus species, sauterium species, verrucomicrobium species, clostridium difficile, clostridium perfringens, coprinus species, dorferia species, enterococcus species, enterobacter sakazakii, clostridium species, proteus species, salmonella species, shigella species, veillonella species, candida albicans, candida tropicalis, candida krusei, candida glabrata; the 5 'end of the probe is marked with a fluorescent group, and the 3' end of the probe is marked with a fluorescent quenching group.
The invention also provides an intestinal flora nucleic acid detection kit, which comprises the set of primers and the probe, and also can comprise an internal control primer for internal control detection:
IC-F1: ATACAATGTATCATGCCTC (namely SEQ ID No.41)
IC-R1: GGCCTAGCTTGGACTCAG (namely SEQ ID No.42)
Or
IC-F2: TGCCTCTTTGCACCATTCT (namely SEQ ID No.43)
IC-R2: GACTCAGAATAATCCAGCC (namely SEQ ID No.44)
And an internal control probe:
IC: TGTAACTGATGTAAGAGGTT, respectively; (i.e., SEQ ID No.45)
All probes are fixed on specific positions of the membrane strip to form a detection chip containing a probe array, and the kit is realized based on a PCR-quantum fluorescence method.
The method for detecting the nucleic acid of the intestinal flora based on the PCR-quantum dot fluorescence method can adopt the kit, and the method comprises the following steps: extracting DNA from a sample as a template, configuring an amplification reaction system, and carrying out PCR amplification; the amplification reaction system is as follows: 13.5. mu.L of water, 2.5. mu.L of 10 XPCR Buffer, 25mM MgCl21.5. mu.L, 25mM dNuTP 0.2. mu.L, 10. mu.M 16SF 10.5. mu.L and 10. mu.M 16SR 10.5. mu.L or 10. mu.M 16SF 20.5. mu.L and 10. mu.M 16SR 20.5. mu.L, 10. mu.M VVC-F20.5. mu.L and 10. mu.M VVC-R20.5. mu.L or 10. mu.M VVC-F10.5. mu.L and 10. mu.M VVC-R10.5. mu.L, 10. mu.M IC-F10.1. mu.L and 10. mu.M IC-R10.1. mu.L or 10. mu.M IC-F20.1. mu.L and 10. mu.M IC-R20.1. mu.L, 1U
0.1 muL, 5U/muL Taq enzyme 1.0 muL; adding 4 mu L of sample, and obtaining 25 mu L of total reaction system; the above is used in one part.
The conditions for performing the PCR amplification were: UNG enzyme reaction at 50 deg.C for 2min, cycle number 1; pre-denaturation at 95 ℃ for 10min, cycle number 1; denaturation 95 ℃ for 30sec, annealing 55 ℃ for 45sec, elongation 72 ℃ for 45sec, cycle number 40; the elongation was then carried out at 72 ℃ for 5min, cycle number 1.
The invention provides a method for comprehensively and rapidly detecting 32 intestinal flora, develops a corresponding kit, and realizes that whether 32 intestinal flora exist or not can be simultaneously obtained by one-time detection for the first time, wherein the 32 intestinal flora contain common probiotics and pathogenic bacteria corresponding to different diseases, and no related technical report exists at present, compared with the prior art, such as: the second generation high-throughput sequencing technology can realize the detection of a large amount of intestinal flora, but can not selectively detect according to different disease requirements, has comprehensive detection data but no emphasis, detects a large amount of bacteria without meaning, and has the advantages of long sequencing period, high cost and complex data analysis.
The invention provides corresponding primers and probes by a method for detecting nucleic acid by PCR-quantum dot fluorescence, can be directly applied to the nucleic acid detection of 32 intestinal flora related to diabetes and obesity, can accurately detect the 32 intestinal flora in one test, and effectively distinguish probiotics and pathogenic bacteria.
Drawings
FIG. 1 shows the results of a plurality of different samples tested by the method of the present invention, (a) sample No.2, (b) sample No.9, and (c) sample No. 31;
Detailed Description
The invention is described in detail below with reference to the figures and examples.
1.1 design and screening of amplification primers
Designing a universal PCR primer pair according to 16S rRNA of bacteria and the evolutionary characteristic sequence thereof; designing a specific PCR primer pair according to a gene sequence between 18S rRNA and 28S rRNA of each candida, and amplifying to obtain a target segment of the intestinal flora with a certain length; in addition, an IC internal control is also designed for monitoring the whole experimental process.
Search and download the 16S rRNA of the bacteria in Genebank database with its evolutionary characteristic sequence,
Designing primers by using primer premier5.0 according to gene sequences between 18S rRNA and 28S rRNA of each candida, wherein Tm values of all amplification primers are similar as much as possible; the designed primers were synthesized by Invitrogen writing. After the primer synthesis, the sequence is checked, and then the primer solution with the required concentration is dissolved and diluted. And screening the detection and typing primers capable of efficiently and stably amplifying through a large number of tests. The change of the length and position of the primer can reduce the sensitivity and the repeatability of the kit, and the obtained primer numbers and sequences are shown in Table 2.
TABLE 2 primer list for detection of intestinal flora
5.2.2 confirmation of PCR amplification reaction System
By utilizing an orthogonal test method and through a large number of experimental comparison optimization, the finally determined PCR reaction system is shown in Table 3, and the amplification efficiency is higher when the system is adopted.
TABLE 3 PCR amplification reaction System recipe
Note: the amount of template added was 4. mu.L, and the total reaction volume was 25. mu.L.
5.2.3 determination of reaction conditions for PCR amplification
After comparative optimization of a large number of experiments, the finally determined PCR amplification reaction conditions are shown in Table 4.
TABLE 4 PCR amplification reaction conditions
5.2.4 design and implementation of probes and Gene chips
According to the difference of different intestinal bacteria genes in 16S rRNA sequences and the difference of candida specific sequences, an oligonucleotide probe for specifically recognizing a certain pathogen sequence and genotype is designed according to the base complementary pairing principle; all oligonucleotide probes are fixed on a specific position of a membrane strip (a glass sheet or a nylon membrane, the basic principles of the glass sheet or the nylon membrane are similar, the preparation process of a glass chip is complex, the detection process is complicated, especially a laser scanner is needed for signal detection, the use cost of the glass chip is high, and the glass chip cannot be effectively popularized in the market, particularly clinical detection.
The design of the probe is designed according to the following principle:
1. the probe length should be between 15-45bp (preferably 20-30bp) as close to the upstream primer as possible to ensure that the DNA folding and secondary structure Tm of the binding specificity detection probe is between 65-70 ℃ and typically 5-10 ℃ higher (at least 5 ℃) than the primer TM, and the GC content is between 40-70%.
2. The content of the base C in the whole probe is obviously higher than that of G, the reaction efficiency is reduced due to the high content of G, and the other paired strand is selected as the probe.
3. To ensure the specificity of the primer probe, it is preferable to check the designed sequence once in blast, and if a non-specific complementary region is found, it is recommended to redesign the primer probe.
The designed probe was synthesized by Invitrogen writing, and the sequence was checked after the probe was synthesized, and then dissolved and diluted to a primer solution of a desired concentration. The probe is fixed on a nylon membrane through the condensation reaction of amino and carboxyl to prepare the detection chip for detecting the intestinal flora. And a stable detection probe with strong specificity is obtained through optimization and screening of a large number of tests. The length and base composition of the probe can affect the specificity and accuracy of the detection of the kit, and the number and sequence of the probe at each site are shown in Table 5. The probe sites on the membrane strip in this example are shown in Table 6.
TABLE 5 Probe sequences and numbering
TABLE 6 Probe site map on Membrane strips
5.2.5 determination of hybridization conditions
Through a series of optimization experiments, the conditions of hybridization, membrane washing, color development and the like are finally determined as follows:
solution A: 100mL of 20 XSSC, 10mL of 10% SDS and 1000mL of purified water were added. And (5) preserving at normal temperature.
And B, liquid B: 25mL of 20 XSSC, 10mL of 10% SDS and 1000mL of purified water were added thereto. And (5) preserving at normal temperature.
QD: quantum dots coupled to streptavidin;
5.2.5.1 hybridization
Taking DNA of a fecal sample as a sample, taking a 5mL six-linked plastic box, placing the six-linked plastic box into a membrane strip marked with a sample number (marked by a gel pen), placing the six-linked plastic box into a hybridization instrument, adding 1mL of solution A, preheating at 48 ℃ for 20 minutes, then adding the two tubes of denatured PCR products with corresponding numbers into the solution A, and hybridizing at 48 ℃ for 1.5 hours.
5.2.5.2 washing membranes
Aspirate solution A, add 1mL of solution B preheated to 48 ℃ and wash with gentle shaking at 48 ℃ for 15 minutes.
5.2.53 fluorescent color development
The formula of the incubation liquid is as follows: solution A: QD 10000:1
Preparing an incubation solution according to the formulation of the incubation solution, sucking out the solution B in the wells, adding 1ml of the incubation solution into each well, incubating for 30 minutes with gentle shaking at room temperature, and sucking out the incubation solution. 1ml of solution A was added to each well, and the membrane was gently shaken at room temperature for 5 minutes. Sucking out the solution A, and observing the result in a fluorescence detector.
According to the distribution of the site maps shown in Table 6, some of the results are shown in FIG. 1, and the results of the detection of sample No.2 are shown in (a), it can be determined that Bifidobacterium pseudocatenulatum, Bifidobacterium longum, Eubacterium, enterococcus, and Shigella are present in sample No. 2; the detection result of sample No.9 (b) can obtain the existence of salmonella and shigella in sample No.9, and the detection result of sample No.31 (c) can indicate the existence of enterococcus and shigella. The results are consistent with the sample case.
Sequence listing
<110> Hangzhou Qianji Biotechnology Co., Ltd
<120> intestinal flora nucleic acid detection kit and detection method based on PCR-quantum dot fluorescence method
<160> 45
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ccggaggaag gtggg 15
<210> 2
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgatccgcga ttactag 17
<210> 3
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggtggggatg acgtca 16
<210> 4
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cgattactag cgattcc 17
<210> 5
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ttgaacgcac attgcgcc 18
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tcctacctga tttgaggtc 19
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
catgcctgtt tgagcgtc 18
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ttgatatgct taagttcag 19
<210> 9
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gcgagggtaa gcggatctc 19
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cgagagtaag ctaatctctt a 21
<210> 11
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
caacgagttg cgagaccg 18
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
acaatggaca gtacaacgag 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
acaatggccg gtacaaagag 20
<210> 14
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ggccggtaca aagagaagc 19
<210> 15
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gcggatccct taaaaccg 18
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
tacctggtga caggatgcta 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
atcccaaaaa cctctctcag 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cctcgcgaga gtgagcaaaa 20
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
caaaactcag aaacaacgtc c 21
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cagagggggc cgaagccgcg a 21
<210> 21
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
caaagagaag cgaaaccgcg a 21
<210> 22
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
aaagtcggat gcccgtaag 19
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tgttaacaga gggaagcaaa a 21
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
cccagaaaac cgatcgtagt 20
<210> 25
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ggaaatcctc aaaactgggc t 21
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gtagtacaga gggttgccaa g 21
<210> 27
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tggtacagag agatgcaata c 21
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gaagcgaagc gcgaggtgga 20
<210> 29
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ctcgcgaggg taagcaaatc t 21
<210> 30
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gaaaccgcga ggtggagcga at 22
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
tgcgtcgtag tccggattgg ag 22
<210> 32
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
agtcgcaaag ctgtgaagtg g 21
<210> 33
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
acctcgcgag agcaagcgga a 21
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
cgacctcgcg agagcaagcg 20
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gcgtcgtagt ccggagggga 20
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
caaaccccag aaacaagctc 20
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gcaatacgac ttgggtttgc 20
<210> 38
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gaatttaacg tggaaactta t 21
<210> 39
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
cgacgtgtaa agagcgtcgg 20
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cagtatgtgg gacacgagcg 20
<210> 41
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
atacaatgta tcatgcctc 19
<210> 42
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
ggcctagctt ggactcag 18
<210> 43
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
tgcctctttg caccattct 19
<210> 44
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gactcagaat aatccagcc 19
<210> 45
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
tgtaactgat gtaagaggtt 20
Claims (5)
1. A set of primers and a probe for detecting nucleic acid of intestinal flora is characterized by comprising amplification primer sequences suitable for 32 intestinal flora detections:
16SF1：CCGGAGGAAGGTGGG
16SR1：TGATCCGCGATTACTAG
or
16SF2：GGTGGGGATGACGTCA
16SR2：CGATTACTAGCGATTCC
And
VVC-F1：TTGAACGCACATTGCGCC
VVC-R1：TCCTACCTGATTTGAGGTC
or
VVC-F2：CATGCCTGTTTGAGCGTC
VVC-R2：TTGATATGCTTAAGTTCAG
And probe sequence from 5 'end to 3' end:
Lbu：GCGAGGGTAAGCGGATCTC
Lre：CGAGAGTAAGCTAATCTCTTA
Lrh：CAACGAGTTGCGAGACCG
Lac：ACAATGGACAGTACAACGAG
Pse：ACAATGGCCGGTACAAAGAG
Lon：GGCCGGTACAAAGAGAAGC
Ado：GCGGATCCCTTAAAACCG
Bth：TACCTGGTGACAGGATGCTA
Bov：ATCCCAAAAACCTCTCTCAG
But：CCTCGCGAGAGTGAGCAAAA
Fae：CAAAACTCAGAAACAACGTCC
Akk：CAGAGGGGGCCGAAGCCGCGA
Eub：CAAAGAGAAGCGAAACCGCGA
Pre：AAAGTCGGATGCCCGTAAG
Rum：TGTTAACAGAGGGAAGCAAAA
Sut：CCCAGAAAACCGATCGTAGT
Ver：GGAAATCCTCAAAACTGGGCT
Cdi：GTAGTACAGAGGGTTGCCAAG
Cpe：TGGTACAGAGAGATGCAATAC
Cop：GAAGCGAAGCGCGAGGTGGA
Dor：CTCGCGAGGGTAAGCAAATCT
EF：GAAACCGCGAGGTGGAGCGAAT
Esa：TGCGTCGTAGTCCGGATTGGAG
Fus：AGTCGCAAAGCTGTGAAGTGG
Pro：ACCTCGCGAGAGCAAGCGGAA
Sal：CGACCTCGCGAGAGCAAGCG
Shi：GCGTCGTAGTCCGGAGGGGA
Vei：CAAACCCCAGAAACAAGCTC
CA：GCAATACGACTTGGGTTTGC
CT：GAATTTAACGTGGAAACTTAT
CK：CGACGTGTAAAGAGCGTCGG
CG：CAGTATGTGGGACACGAGCG
the corresponding 32 intestinal flora are in turn: lactobacillus bulgaricus, lactobacillus reuteri, lactobacillus rhamnosus, lactobacillus acidophilus, bifidobacterium pseudocatenulatum, bifidobacterium longum, bifidobacterium adolescentis, bacteroides multiradiae, bacteroides ovatus, clostridium butyricum, clostridium pratensis, eckmann species, eubacterium species, prevotella, ruminococcus species, sauterium species, verrucomicrobium species, clostridium difficile, clostridium perfringens, coprinus species, dorferia species, enterococcus species, enterobacter sakazakii, clostridium species, proteus species, salmonella species, shigella species, veillonella species, candida albicans, candida tropicalis, candida krusei, candida glabrata; the 5 'end of the probe is marked with a fluorescent group, and the 3' end of the probe is marked with a fluorescent quenching group.
2. An intestinal flora nucleic acid detection kit, comprising the set of primers and the probe of claim 1.
3. The intestinal flora nucleic acid detection kit of claim 2, wherein,
also comprises an internal control primer for internal control detection:
IC-F1：ATACAATGTATCATGCCTC
IC-R1：GGCCTAGCTTGGACTCAG
or
IC-F2：TGCCTCTTTGCACCATTCT
IC-R2：GACTCAGAATAATCCAGCC
And an internal control probe:
IC：TGTAACTGATGTAAGAGGTT；
all probes are fixed on specific positions of the membrane strip to form a detection chip containing a probe array, and the kit is realized based on a PCR-quantum fluorescence method.
4. A method for detecting nucleic acid of intestinal flora based on a PCR-quantum dot fluorescence method is characterized in that the kit of claim 3 is adopted, and the method comprises the following steps: extracting DNA from a sample as a template, configuring an amplification reaction system, and carrying out PCR amplification; the amplification reaction system is as follows: 13.5. mu.L of water, 2.5. mu.L of 10 XPCR Buffer, 25mM MgCl21.5. mu.L, 25mM dNuTP 0.2. mu.L, 10. mu.M 16SF 10.5. mu.L and 10. mu.M 16SR 10.5. mu.L or 10. mu.M 16SF 20.5. mu.L and 10. mu.M 16SR 20.5. mu.L, 10. mu.M VVC-F20.5. mu.L and 10. mu.M VVC-R20.5. mu.L or 10. mu.M VVC-F10.5. mu.L and 10. mu.M VVC-R10.5. mu.L, 10. mu.M IC-F10.1. mu.L and 10. mu.M IC-R10.1. mu.L or 10. mu.M IC-F20.1. mu.L and 10. mu.M IC-R20.1. mu.L, 1. mu.L of UNG enzyme 0.1. mu.L, 5U/. mu; adding 4 mu L of sample, and obtaining 25 mu L of total reaction system; the above is used in one part.
5. The method for detecting nucleic acid in intestinal flora based on PCR-quantum dot fluorescence method of claim 4, wherein the PCR amplification is performed under the following conditions: UNG enzyme reaction at 50 deg.C for 2min, cycle number 1; pre-denaturation at 95 ℃ for 10min, cycle number 1; denaturation 95 ℃ for 30sec, annealing 55 ℃ for 45sec, elongation 72 ℃ for 45sec, cycle number 40; the elongation was then carried out at 72 ℃ for 5min, cycle number 1.
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