CN112899339B - Application of streptomycete aureofaciens as marker for detecting and diagnosing hypertension to preparation of corresponding detection tool - Google Patents

Application of streptomycete aureofaciens as marker for detecting and diagnosing hypertension to preparation of corresponding detection tool Download PDF

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CN112899339B
CN112899339B CN202110274048.1A CN202110274048A CN112899339B CN 112899339 B CN112899339 B CN 112899339B CN 202110274048 A CN202110274048 A CN 202110274048A CN 112899339 B CN112899339 B CN 112899339B
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hypertension
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CN112899339A (en
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马鑫
周婷婷
翟齐啸
王志玮
郭梦婷
刘靓靓
肖望
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Jiangnan University
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Abstract

The invention discloses application of Streptomyces aureofaciens Tu 117 serving as a marker for detecting and diagnosing hypertension to prepare a corresponding detection tool, wherein the detection object of the detection tool is feces, intestinal tissues or intestinal contents collected from a subject. The Streptomyces aureofaciens Tu 117 has relatively high content in the bodies of a hypertension model mouse and a hypertension patient, can be used for establishing a detection method or preparing a detection and diagnosis kit, and is used for auxiliary diagnosis and risk assessment of hypertension and pre-hypertension.

Description

Application of streptomycete aureofaciens as marker for detecting and diagnosing hypertension to preparation of corresponding detection tool
Technical Field
The invention relates to a biomarker, in particular to a streptomycete Tu 117 biomarker in feces and application thereof.
Background
Cardiovascular diseases such as hypertension are the leading cause of harm to human health and death. The prevalence of hypertension tends to increase year by year. The Chinese hypertension investigation (Status of hypertension in China: results from the China Hypertension Survey,2012-2015.Circulation,2018;137 (22): 2344-2356) adopts a layered, multi-stage and random sampling method to extract 451755 residents which are more than or equal to 18 years old from 262 cities and rural areas in 31 provinces in China in 2012-2015, and the result shows that the prevalence rate of Chinese adult hypertension is 27.9%, the prevalence rate of male is higher than that of female (28.6% vs. 27.2%), and the prevalence rate is increased with age.
Although the increased blood pressure level is the most important clinical manifestation of hypertension, it is not necessarily the only and desirable diagnostic criteria. A variety of metabolic disorders are often incorporated during the development of hypertension. The human intestinal flora is involved in the digestion of food mainly through glycogenolysis and proteolytic metabolic pathways. The intestinal tract is taken as a excretory organ of main metabolites of the organism, and if the metabolites cannot be effectively removed in time, each organ of the organism will be affected and the heart cannot be avoided. Trimethylamine (TMA) is an organic compound produced by intestinal flora and is metabolized by the liver to form trimethylamine oxide (TMA) which has an atherogenic effect, and increased plasma TMA levels, giant foam cell formation, and increased aortic atherosclerotic plaque size are found in mice on a choline-enriched diet (Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, proteins atheroclysis. Nat. Med.2013;19 (5): 576-585). In addition, intestinal microorganisms can break down phenylalanine-producing metabolic byproducts, phenylacetylglutamine (PAGln), which are rich in plant and animal derived proteins, and the content of PAGln in plasma is associated with cardiovascular disease and major cardiovascular adverse events, such as myocardial infarction, stroke or death (ACardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic receptors. Cell;2020;180, 862-877). Changes in the living environment of the human body or changes in the disease state are usually accompanied by changes in the symbiotic microorganisms in the body, and changes in the metabolism of symbiotic microorganisms are usually earlier than the clinical manifestations of the disease, so that the core symbiotic microorganisms or intestinal microorganisms associated with the disease will be potential biomarkers, which have good auxiliary effects on early diagnosis of the course of the disease. In clinical work, the marker analysis is considered, high risk groups are identified early, and glycolipid metabolism and the like are actively controlled through lifestyle intervention early, so that the occurrence of an acute event is reduced.
Through investigation, no relevant literature report (from PUBMED, CNKI, and Viep journal net) on the correlation of microorganism Streptomyces aureofaciens Tu 117 and hypertension is found by scholars at home and abroad, so that the intestinal tract fungus Streptomyces aureofaciens Tu 117 is still in a blank stage in the field of research on biomarkers of hypertension.
Disclosure of Invention
Aiming at the problems existing in the prior art, the applicant provides an application of preparing a corresponding detection tool by taking streptomyces aureofaciens as a marker for detecting and diagnosing hypertension. The Streptomyces aureofaciens Tu 117 provided by the invention is used as a marker to be associated with the generation and development of metabolites or intestinal bacteria of organisms and hypertension, and has a good auxiliary effect on early diagnosis of the disease course.
The technical scheme of the invention is as follows:
the application of Streptomyces aureofaciens Tu 117 as a marker for detecting and diagnosing hypertension to prepare a corresponding detection tool, wherein the detection object of the detection tool is a sample to be detected collected from a subject; the sample to be tested is feces, intestinal tissue or intestinal contents.
The 16S rDNA sequence of the Streptomyces aureofaciens Tu 117 is shown in SEQ No. 1.
When the relative content of Streptomyces aureofaciens Tu 117 in the feces of the subject is above 1.52, the subject is likely to suffer from hypertension.
When used for the auxiliary diagnosis of hypertension, it comprises the following steps:
(1) Determining the relative content of a marker namely Streptomyces aureofaciens Tu 117 in a sample to be tested of a subject;
(2) Comparing the relative content obtained in the step (1) with a reference value;
(3) When the relative content of S.aureofaciens Tu 117 in the test sample of the subject is higher than the reference value, the subject has a possibility of suffering from hypertension.
In the step (2), the reference value is the relative content of Streptomyces aureofaciens Tu 117 of healthy people, and the relative content is 0.44-1.52.
The hypertension is a hypertension disease in which the systolic pressure of a human body is more than or equal to 140 mmHg and the diastolic pressure is more than or equal to 90 mmHg.
A method of detecting a marker of said marker for non-diagnostic purposes, said method comprising the steps of:
(1) Extracting total RNA of a sample to be tested of a subject, and measuring concentration and absorbance;
(2) Reverse transcribing the RNA obtained in step (1) into cDNA;
TABLE 1 reverse transcription System
The reaction procedure for reverse transcription is: 15min at 37 ℃; 5s at 85 ℃;4 ℃ is infinity.
(3) Amplifying the cDNA obtained in the step (2) on a fluorescent real-time quantitative PCR instrument;
TABLE 2 q PCR amplification System
The PCR reaction procedure was: pre-denaturation at 95℃for 30s; amplifying the mixture at 95 ℃ for 5 seconds, 60 ℃ for 30 seconds and 40cycles; melting curves of 95 ℃ for 5s,60 ℃ for 60s,95 ℃ for 5s and 1cycle; cooling to 55 deg.c for 30s and 1cycle. The primers used for PCR are shown in the following table.
TABLE 3 q-PCR primer sequences
(4) 2 by melting Curve and amplification Curve analysis -△△Ct The method was used for relative quantification.
The detection tool for the hypertension is a diagnostic reagent, a chip, a carrier or a kit.
A method of collecting data of stool, intestinal tissue or intestinal content for aiding in the determination of hypertension, comprising determining the amount of streptomyces aureofaciens Tu 117 in the stool, intestinal tissue or intestinal content collected from a subject who is not clear of whether or not it is suffering from hypertension or a subject whose period of hypertension is not determined.
The beneficial technical effects of the invention are as follows:
the invention indicates for the first time that the content of Streptomyces aureofaciens Tu 117 in a hypertensive model mouse and a hypertensive patient is relatively high, and that the Streptomyces aureofaciens Tu 117 is of significance in distinguishing the hypertensive patient from the healthy population. AUC values of 0.933, 95% CI of 0.868-0.999, sensitivity at the optimal critical point of 84.6% and specificity of 100% were used to distinguish hypertensive and healthy persons using Tu 117. Can be used for establishing a detection method or preparing a diagnosis kit for auxiliary diagnosis of hypertension and pre-hypertension.
Drawings
FIG. 1 shows the relative content of S.aureofaciens Tu 117 in faeces of normal mice and hypertensive mice induced by a high salt diet.
FIG. 2 shows the relative content of S.aureofaciens Tu 117 in feces of healthy people and hypertensive patients.
FIG. 3 is a ROC curve of Streptomyces aureofaciens Tu 117 in feces distinguishing healthy and hypertensive subjects.
FIG. 4 shows the blood pressure of Streptomyces aureofaciens Tu 117 after lavage.
Detailed Description
The present invention will be described in detail below with reference to the drawings and examples.
The marker related to hypertension provided by the invention is Streptomyces aureofaciens Tu 117.
Animal, laboratory instrument and reagent sources related to the specific embodiments:
c57BL/6J mice: shanghai Laike laboratory animal Limited
Sterile C57BL/6J mice: nanjing Jizhikang biological Co.Ltd
Noninvasive blood pressure test apparatus: U.S. IITC
Centrifuge: thermo Fisher
Fluorescent real-time quantitative PCR instrument: roche in the United states
Turbidimeter of mahalanobis: solarbio
High-salt feed: nantong Telofei feed technology Co.Ltd
Reverse transcriptase, q-PCR enzyme: taKaRa
Fecal RNA extraction kit: changzhou Baidai
The culture medium comprises the following components: solarbio
Example 1: high salt diet induced high levels of S.aureofaciens Tu 117 in hypertensive mice
Extracting total RNA of feces of 7 high-salt diet induced hypertension C57BL/6J mice and 6 normal diet control group C57BL/6J mice, and measuring concentration and absorbance; reverse transcribing the obtained RNA into cDNA; amplifying the obtained cDNA on a fluorescent real-time quantitative PCR instrument; the primers required for amplification consisted of forward and reverse primer sequences of the Streptomyces aureofaciens Tu 117-specific gene LipReg4 (Horbal L, rebets Y, rabyk M, luzhetsky A, ostash B, welle E, nakamura T, fedorenko V, bechthold A. Characterization and analysis of the regulatory network involved in control of lipomycin biosynthesis in Streptomyces aureofaciens T u 117.Applied microbiology and biotechnology.2010;85:1069-1079), the forward primer sequences being: 5'-TGGATTTGCTCATCGAGAGG-3', the reverse primer sequence is: 5'-TTCTGCCGTTTCCGGCTCTC-3'.
The Ct value of each sample is obtained through a fluorescent quantitative PCR experiment, and the variation formula of the expression multiple of the fecal mRNA of the hypertension group relative to the control group sample is RQ=2 -△△Ct Calculating the relative content of Streptomyces aureofaciens Tu 117 in different samples, wherein DeltaCt=the Ct value of the target gene-Ct value of the internal reference gene, deltaCt= -DeltaCt Hypertension of the type -△Ct Control 。
As a result, as shown in FIG. 1, the relative content of Streptomyces aureofaciens Tu 117 in the feces of the mice of the control group was 1.00217 + -0.05566, the relative content of Streptomyces aureofaciens Tu 117 in the feces of the hypertensive group was 1.46657 + -0.04086, and the content of Streptomyces aureofaciens Tu 117 in the feces of the hypertensive group was significantly increased, compared with the feces of the control group, over 0.46 times the content of the feces of the control group, and the difference was statistically significant (P < 0.0001). The results demonstrate that the levels of S.aureofaciens Tu 117 were higher in high salt diet induced hypertensive mice.
Example 2: the Streptomyces aureofaciens Tu 117 level in the hypertensive population is higher
The clinical trial recruited 104 volunteers altogether, 26 of the healthy population and 78 of the hypertensive population. All volunteers signed informed consent, all volunteers met inclusion and exclusion criteria. The criteria are as follows:
inclusion criteria: hypertension is a healthy population or clinically diagnosed.
Exclusion criteria: (1) known or suspected as secondary hypertension; (2) Suffering from severe chronic diseases (including heart failure, renal failure, lung disease, rheumatism, stroke, gastrointestinal diseases or hematological diseases, etc.); (3) Antibiotic or probiotic treatment was received over nearly 1 month; (4) suffering from severe cardiovascular disease and related surgical history; (5) A tonic/drug having gastrointestinal inflammation or taking prescribed anti-inflammatory drugs or possibly affecting inflammation; (6) Undergo cholecystectomy, intestinal resection or gastrectomy surgery; (7) malignancy; (8) gestational women; (9) drug use allergy sufferers; (10) researchers consider unsuitable for panelists.
Collecting fecal samples of 78 patients with hypertension and 26 healthy people, extracting total RNA of the feces, and measuring the concentration and absorbance; reverse transcribing the obtained RNA into cDNA; amplifying the obtained cDNA on a fluorescent real-time quantitative PCR instrument; the primer needed for amplification consists of a forward primer sequence and a reverse primer sequence; for S.aureofaciens Tu 117, the forward primer sequences were:
5'-TGGATTTGCTCATCGAGAGG-3', the reverse primer sequence is:
5’-TTCTGCCGTTTCCGGCTCTC-3’。
the Ct value of each sample is obtained through a fluorescent quantitative PCR experiment, and the variation formula of the expression multiple of the fecal mRNA of the hypertensive population relative to the healthy population sample is RQ=2 -△△Ct Calculating the relative content of Streptomyces aureofaciens Tu 117 in different populations, wherein DeltaCt=the Ct value of the target gene-Ct value of the internal reference gene, deltaCt= -DeltaCt Hypertension crowd -△Ct Healthy people 。
As shown in FIG. 2, the relative content of Streptomyces aureofaciens Tu 117 in the feces of normal human beings was 1.019.+ -. 0.04179, and the content of Streptomyces aureofaciens Tu 117 in the feces of patients with hypertension was 10.34.+ -. 2.444. Compared with healthy people, the content of streptomyces aureofaciens Tu 117 in the faeces of hypertensive patients is obviously increased, which is 9.15 times higher than that of healthy people, and the difference has statistical significance (P < 0.0001). The results demonstrate that the levels of S.aureofaciens Tu 117 are higher in the hypertensive population.
Example 3: evaluation of diagnostic Effect of Streptomyces aureofaciens Tu 117 on patients with hypertension
Based on the determined relative levels of S.aureofaciens Tu 117 in fecal samples, tu 117 was analyzed for correlation with hypertensive patients using one-way logistic regression, and the results are shown in the following table, indicating a strong correlation between Tu 117 and hypertensive patients.
TABLE 4 Single factor logistic regression analysis results
According to 2 -△△Ct Calculating the relative expression quantity of Tu 117 in the feces, and carrying out regression analysis by using SPSS software to obtain the formula: logit (P) = 4.077 ×LipReg4-5.787. Substituting the detected relative expression amount of LipReg4 into the above equation, if the calculated value is higher than 0.41, diagnosing hypertension, and if the calculated value is lower than 0.41, judging non-hypertension.
In order to evaluate the ability of Tu 117 to detect hypertension, the present invention performs risk assessment on the fluorescent quantitative PCR results of the control group of hypertension and healthy people, and evaluates the value of Streptomyces aureofaciens Tu 117 in hypertension diagnosis by ROC. And (3) sensitivity is taken as an ordinate to represent true positive rate, 1-specificity is taken as an abscissa to represent false positive rate, and ROC curve analysis is performed. The larger the area under the curve (AUC), the higher the diagnostic accuracy. On the ROC curve, the point closest to the upper left of the graph is the critical value for both higher sensitivity and specificity.
The AUC value of the Tu 117 was 0.933, the 95% CI was 0.868-0.999, the sensitivity at the optimal critical point was 84.6% and the specificity was 100% when used to distinguish between hypertensive and healthy persons (FIG. 3), which indicated that Tu 117 was of interest in distinguishing between hypertensive and healthy persons.
Example 4: streptomyces aureofaciens Tu 117 stomach lavage for increasing blood pressure of mice
Taking 16 sterile C57BL/6J mice of 6-8 weeks old, feeding into a sterile bin, randomly dividing into two groups, which are respectively experimental groups [ (]Streptomyces aureofaciens Tu 117 strain lavage) and control group (saline lavage). Streptomyces aureofaciens Tu 117 strain was cultured and amplified using International Streptomyces project-1 (ISP-1) liquid medium in an aerobic environment at 28℃and shaking culture at 220rpm for 2 days until a significantly dense granular cell floating was seen in the medium. Centrifuging to collect thallus, counting with Mitsubishi turbidimeter, and mixing thallus with physiological saline to obtain thallus with concentration of 10 8 CFU/mL bacterial liquid. The bacteria liquid is irrigated into the stomach of a sterile mouse, and 300 mu L of each mouse is irrigated into the stomach for 7 times in two days, and the control group is irrigated with physiological saline with the same volume. On day 16, the mice were taken from the sterile warehouse and tested for blood pressure using the tail boot method on a sterile console.
14C 57BL/6J mice of 6-8 weeks old were housed in specific pathogen-free (Specific pathogen Free, SPF) class barriers and randomly divided into two groups, an experimental group (S.aureofaciens Tu 117 strain lavage) and a control group (saline lavage). Preparing the thallus into 10 8 CFU/mL lavage mice, 300 μl each, were lavaged once daily for 9 weeks, and control group was lavaged with equal volumes of saline. Mice were monitored weekly for blood pressure changes using the tail jacket method.
As shown in fig. 4, after 7 times of intragastric administration of the sterile mice, the systolic blood pressure of the control group was 90.375 ± 1.76208mmHg, and the systolic blood pressure of the experimental group was 109.625 ± 1.91738mmHg, indicating that the blood pressure of the mice showed a significant increase, 19mmHg higher than that of the control group, and had a statistical significance (P < 0.0001) (see fig. 4A). After the normal mice raised in SPF-grade were perfused with Streptomyces aureofaciens Tu 117, the blood pressure of the mice increased from the second week, and the blood pressure was substantially stabilized at a higher level in the fourth week, and the results are shown in Table 5; the differences were significant (< P < 0.0001) compared to the control mice group (fig. 4B). The results demonstrate that Streptomyces aureofaciens Tu 117 perfuses the stomach to cause hypertension in mice.
TABLE 5 weekly blood pressure changes after SPF-grade feeding of normal mice with Streptomyces aureofaciens Tu 117
The Streptomyces aureofaciens Tu 117 can be applied to preparing a corresponding detection tool as a marker for detecting and diagnosing hypertension. The development and the application of the intestinal microorganism streptomycete aureobasidium 117 biomarker in the detection kit enable the detection of hypertension and the early stage of hypertension to be convenient and easy, and provide support for clinicians to quickly find the early stage of hypertension and timely adopt an effective prevention and treatment scheme.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
SEQUENCE LISTING
<110> university of Jiangnan
<120> application of Streptomyces aureofaciens as marker for detecting and diagnosing hypertension to prepare corresponding detection tool
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1380
<212> DNA
<213> Streptomyces aureofaciens Tu 117 16srDNA
<400> 1
tgcaagtcga acgatgaacc acttcggtgg ggattagtgg cgaacgggtg agtaacacgt 60
gggcaatctg ccctgcactc tgggacaagc cctggaaacg gggtctaata ccggatactg 120
accctcgcag gcatctgcga ggttcgaaag ctccggcggt gcaggatgag cccgcggcct 180
atcagcttgt tggtgaggta atggctcacc aaggcgacga cgggtagccg gcctgagagg 240
gcgaccggcc acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg 300
aatattgcac aatgggcgaa agcctgatgc agcgacgccg cgtgagggat gacggccttc 360
gggttgtaaa cctctttcag cagggaagaa gcgaaagtga cggtacctgc agaagaagcg 420
ccggctaact acgtgccagc agccgcggta atacgtaggg cgcaagcgtt gtccggaatt 480
attgggcgta aagagctcgt aggcggcttg tcgcgtcggt tgtgaaagcc cggggcttaa 540
ccccgggtct gcagtcgata cgggcaggct agagttcggt aggggagatc ggaattcctg 600
gtgtagcggt gaaatgcgca gatatcagga ggaacaccgg tggcgaaggc ggatctctgg 660
gccgatactg acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta 720
gtccacgccg taaacggtgg gcactaggtg tgggcaacat tccacgttgt ccgtgccgca 780
gctaacgcat taagtgcccc gcctggggag tacggccgca aggctaaaac tcaaaggaat 840
tgacgggggc ccgcacaagc ggcggagcat gtggcttaat tcgacgcaac gcgaagaacc 900
ttaccaaggc ttgacataca ccggaaagca tcagagatgg tgcccccctt gtggtcggtg 960
tacaggtggt gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1020
cgagcgcaac ccttgtcccg tgttgccagc aggcccttgt ggtgctgggg actcacggga 1080
gaccgccggg gtcaactcgg aggaaggtgg ggacgacgtc aagtcatcat gccccttatg 1140
tcttgggctg cacacgtgct acaatggccg gtacaatgag ctgcgatacc gcaaggtgga 1200
gcgaatctca aaaagccggt ctcagttcgg attggggtct gcaactcgac cccatgaagt 1260
cggagtcgct agtaatcgca gatcagcatt gctgcggtga atacgttccc gggccttgta 1320
cacaccgccc gtcacgtcac gaaagtcggt aacacccgaa gccggtggcc caaccccttg 1380

Claims (6)

1. The application of preparing a corresponding detection tool by taking Streptomyces aureofaciens Tu 117 as a marker for detecting and diagnosing hypertension is characterized in that the detection object of the detection tool is a sample to be detected collected from a subject; the sample to be tested is feces.
2. The use according to claim 1, characterized in that the 16S rDNA sequence of S.aureofaciens Tu 117 is shown in SEQ No. 1.
3. The use according to claim 1, wherein a relative content of streptomyces aureofaciens Tu 117 in the faeces of the subject of more than 1.52 indicates a likelihood of the subject having a hypertensive disorder; the relative content is relative to a reference gene, and the sequence of an upstream primer of the reference gene is 5'-ACTCCTACGGGAGGCAGCAG-3'; the sequence of the downstream primer was 5'-ATTACCGCGGCTGCTGG-3'.
4. The use according to claim 1, characterized in that it comprises the following steps when used for the auxiliary diagnosis of hypertension:
(1) Determining the relative content of a marker namely Streptomyces aureofaciens Tu 117 in a sample to be tested of a subject;
(2) Comparing the relative content obtained in the step (1) with a reference value;
(3) When the relative content of Streptomyces aureofaciens Tu 117 in a sample to be tested of a subject is higher than a reference value, the subject has a possibility of suffering from hypertension;
the relative content is relative to a reference gene, and the sequence of an upstream primer of the reference gene is 5'-ACTCCTACGGGAGGCAGCAG-3'; the sequence of the downstream primer was 5'-ATTACCGCGGCTGCTGG-3'.
5. The use according to claim 4, wherein in step (2) the reference value is the relative content of Streptomyces aureofaciens Tu 117 of a healthy population, which is 0.44-1.52.
6. The use according to claim 1, wherein the hypertension is a disease in which a systolic blood pressure of 140 mmhg or more and a diastolic blood pressure of 90 mmhg or more are detected.
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