CN116515992A - Application of reagent for detecting vaginal microorganism abundance in preparation of premature labor and prematurity auxiliary diagnosis kit - Google Patents
Application of reagent for detecting vaginal microorganism abundance in preparation of premature labor and prematurity auxiliary diagnosis kit Download PDFInfo
- Publication number
- CN116515992A CN116515992A CN202310704577.XA CN202310704577A CN116515992A CN 116515992 A CN116515992 A CN 116515992A CN 202310704577 A CN202310704577 A CN 202310704577A CN 116515992 A CN116515992 A CN 116515992A
- Authority
- CN
- China
- Prior art keywords
- vaginal
- pregnant women
- premature
- reagent
- detecting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 244000005700 microbiome Species 0.000 title claims abstract description 18
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 11
- 206010036590 Premature baby Diseases 0.000 title claims abstract description 10
- 208000006399 Premature Obstetric Labor Diseases 0.000 title abstract description 45
- 206010036600 Premature labour Diseases 0.000 title abstract description 37
- 208000026440 premature labor Diseases 0.000 title abstract description 37
- 238000003745 diagnosis Methods 0.000 title abstract description 6
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 206010036595 Premature delivery Diseases 0.000 claims abstract description 10
- 241000736131 Sphingomonas Species 0.000 claims description 11
- 241000122971 Stenotrophomonas Species 0.000 claims description 10
- 241000863012 Caulobacter Species 0.000 claims description 8
- 241000607715 Serratia marcescens Species 0.000 claims description 8
- 241001135223 Prevotella melaninogenica Species 0.000 claims description 6
- 241000590020 Achromobacter Species 0.000 claims description 5
- 241000605861 Prevotella Species 0.000 claims description 4
- 241000607720 Serratia Species 0.000 claims description 4
- 239000000523 sample Substances 0.000 claims description 4
- 241000186660 Lactobacillus Species 0.000 claims description 2
- 229940039696 lactobacillus Drugs 0.000 claims description 2
- 230000003061 melanogenesis Effects 0.000 claims description 2
- 241000023308 Acca Species 0.000 claims 1
- 238000009007 Diagnostic Kit Methods 0.000 claims 1
- 241001671204 Stemona Species 0.000 claims 1
- 239000000853 adhesive Substances 0.000 claims 1
- 230000001070 adhesive effect Effects 0.000 claims 1
- 230000035935 pregnancy Effects 0.000 abstract description 13
- 241000894006 Bacteria Species 0.000 abstract description 9
- 230000000813 microbial effect Effects 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 5
- 238000013399 early diagnosis Methods 0.000 abstract description 5
- 238000012163 sequencing technique Methods 0.000 abstract description 5
- 208000005107 Premature Birth Diseases 0.000 abstract description 4
- 239000000090 biomarker Substances 0.000 abstract description 4
- 230000002411 adverse Effects 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
- 230000009897 systematic effect Effects 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 description 11
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 8
- 241000566145 Otus Species 0.000 description 7
- 230000002028 premature Effects 0.000 description 7
- 241000736262 Microbiota Species 0.000 description 6
- 208000004926 Bacterial Vaginosis Diseases 0.000 description 5
- 208000037009 Vaginitis bacterial Diseases 0.000 description 5
- 210000001215 vagina Anatomy 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 241000207202 Gardnerella Species 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 208000037805 labour Diseases 0.000 description 4
- 235000014655 lactic acid Nutrition 0.000 description 4
- 239000004310 lactic acid Substances 0.000 description 4
- 230000001681 protective effect Effects 0.000 description 4
- 229920000742 Cotton Polymers 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 238000000585 Mann–Whitney U test Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000010239 partial least squares discriminant analysis Methods 0.000 description 3
- 108091093088 Amplicon Proteins 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 241000192142 Proteobacteria Species 0.000 description 2
- 208000036029 Uterine contractions during pregnancy Diseases 0.000 description 2
- 206010000210 abortion Diseases 0.000 description 2
- 231100000176 abortion Toxicity 0.000 description 2
- 210000004381 amniotic fluid Anatomy 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 210000003756 cervix mucus Anatomy 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 238000000513 principal component analysis Methods 0.000 description 2
- 230000000750 progressive effect Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 230000036266 weeks of gestation Effects 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 208000005952 Amniotic Fluid Embolism Diseases 0.000 description 1
- 206010067010 Anaphylactoid syndrome of pregnancy Diseases 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 208000034702 Multiple pregnancies Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 208000002787 Pregnancy Complications Diseases 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 208000035010 Term birth Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 210000001691 amnion Anatomy 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000010339 dilation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000002219 extraembryonic membrane Anatomy 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 210000005002 female reproductive tract Anatomy 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 230000007373 microbial translocation Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 208000012113 pregnancy disease Diseases 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 238000012175 pyrosequencing Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000010845 search algorithm Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/425—Serratia
- C12R2001/43—Serratia marcescens
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses application of a reagent for detecting vaginal microorganism abundance in preparation of a premature labor auxiliary diagnosis kit. Belongs to the technical field of biological medicine. The invention adopts the microbial diversity to study the vaginal microenvironment of full-term delivery, premature delivery and pre-term delivery pregnant women. Screening for differential bacteria between the three groups by 16SrRNA sequencing demonstrates the potential of the flora to develop a systematic model to understand the vaginal environment for risk of premature birth. The biomarker with excellent diagnostic capability is screened out, and can be used for daily prenatal examination of pregnant women, as an early diagnosis means for pregnant women with premature labor risk or pre-premature labor, improve the pregnancy ending of the pregnant women, improve the pregnancy quality of the pregnant women and reduce the occurrence of adverse events of newborns.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to application of a reagent for detecting vaginal microorganism abundance in preparation of a premature labor auxiliary diagnosis kit.
Background
Pre-term labor refers to regular or irregular uterine contractions at 28 weeks but less than 37 weeks of gestation with progressive shortening of the cervical canal and cervical dilation of less than 1cm. The pre-term pregnant women are frequently in emergency hospitalization, have uncertainty on the premature delivery, are easy to anxiety and depression, and influence the pregnancy quality. Meanwhile, premature infants may develop weaker body and whole body than full term infants. Therefore, it is important to diagnose abnormal pregnancy of pregnant women as soon as possible, improve pregnancy outcome and improve miscarriage prevention success rate. There are many factors responsible for pre-term labour, of which infection is an important contributor to pre-term labour. During pregnancy, pathogens rise from the vagina into the uterine cavity, amniotic membrane, and even spread into amniotic fluid, resulting in premature labor or abortion. In recent years, many studies have focused on the impact of vaginal microbiota/microbiome on premature delivery, but there is no clear understanding of its pathophysiology, nor has effective biomarkers and clinical interventions been found to improve pregnancy outcome.
Under normal conditions, various microorganisms inhabit in the vagina of a female, form a dynamic balance relation which is mutually restricted and coordinated with a host and the environment, and mainly use lactic acid bacteria to maintain the acidic environment of the vagina. However, the relationship between imbalances in the vaginal bacterial community, bacterial Vaginosis (BV) and premature labor is widely reported. Vaginal microecological balance plays a key role in maintaining maternal health and fetal normal development.
In summary, how to provide a vaginal microbial marker for assisting in diagnosing premature labor and prematurity is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of this, the invention provides the use of a reagent for detecting vaginal microbial abundance in the preparation of a kit for assisting diagnosis of preterm birth and prematurity.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
use of a reagent for detecting abundance of a vaginal microorganism in the preparation of a kit for assisting diagnosis of premature delivery and prematurity, wherein the vaginal microorganism is one or more of Prevotella melanogenesis (Prevotella melaninogenica), serratia viscosa (Serratia marcescens), stenotrophomonas (stenotrophomonas), bacillus (Caulobacter), sphingomonas (Sphingomonas), and Achromobacter (Achromobacter agglutinosus).
Further, the reagent for detecting the abundance of the vaginal microorganisms is a primer or a probe for detecting the vaginal microorganisms.
Further, the reagent for detecting the abundance of vaginal microorganisms is a primer for detecting Prevotella melanogensis (Prevotella melaninogenica), serratia viscosa (Serratia marcescens), stenotrophomonas (stenotrophomonas), bacillus (Caulobacter), sphingomonas (Sphingomonas), and Achromobacter (Lidocarpus) 16SrRNA.
Compared with the prior art, the invention has the beneficial effects that: the invention adopts the microbial diversity to study the vaginal microenvironment of full-term delivery, premature delivery and pre-term delivery pregnant women. Screening for differential bacteria between the three groups by 16S rRNA sequencing demonstrates the potential of the flora to develop a systematic model to understand the vaginal environment for risk of premature delivery. The biomarker with excellent diagnostic capability is screened out, and can be used for daily prenatal examination of pregnant women, as an early diagnosis means for pregnant women with premature labor risk or pre-premature labor, improve the pregnancy ending of the pregnant women, improve the pregnancy quality of the pregnant women and reduce the occurrence of adverse events of newborns.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a summary of the classification of intestinal flora of groups FB, PB and TPB at the (A) phylum level and the (B) genus level in example 1 of the present invention;
FIG. 2 is an OTUs observed in the analysis of the diversity of the groups FB, TPB and PB in example 1 of the present invention, wherein x represents p <0.05 by Mann-Whitney U test;
FIG. 3 shows the results of Chao1 for groups FB, TPB and PB in example 1 of the present invention, where p <0.05 is represented by Mann-Whitney U test;
FIG. 4 shows the results of the groups FB, TPB and PB of Shannon in example 1 of the present invention;
FIG. 5 shows Simpson results for groups FB, TPB and PB in example 1 of the present invention;
FIG. 6 shows the results of the PCoA groups FB, TPB and PB in example 1 of the present invention;
FIG. 7 shows the result of the PLS-DA for groups FB, TPB and PB in example 1 of the present invention;
fig. 8 is a branching diagram (FB and PB) of the LEfSe method used in embodiment 1 of the present invention;
FIG. 9 shows LDA scores (FB and PB) in example 1 of the present invention;
fig. 10 is a branching diagram (PB and TPB) of the LEfSe method used in example 1 of the present invention;
FIG. 11 shows LDA scores (PB and TPB) in example 1 of the present invention;
FIG. 12 shows the relative abundance of Prevotella melaninogenica in three groups of participants in an embodiment of the invention;
FIG. 13 is a graph showing the relative abundance of Stenotrophomonas in three groups of participants in the examples of the invention;
FIG. 14 shows the relative abundance of Caulobacter among three groups of participants in the examples of the invention;
FIG. 15 is a graph showing the relative abundance of Sphingomonas among three groups of participants in the examples of the present invention;
fig. 16 is a graph of relative abundance of Serratia marcescens in three groups of participants in an embodiment of the invention;
FIG. 17 is a graph showing the relative abundance of the Lidocacaulis in three groups of participants in the examples of the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The required medicament is a conventional experimental medicament and is purchased from a commercial channel; the test methods not mentioned are conventional test methods and will not be described in detail herein.
Example 1
1. Method of
1.1 patient and public participation
84 pregnant women participated in this study.
1.2 subject and sample collection
Vaginal secretions were collected at the university of Zhongshan affiliated eighth hospital from 7 months 2018 to 11 months 2018.
Inclusion criteria for pregnant women included:
1) Age 18-48 years.
2) The women with prematurity meet the diagnostic standard: the fetal membranes are complete, the opening of the uterine opening is less than 1cm, the irregular uterine contractions (20 minutes is more than or equal to 4 times or 60 minutes is more than or equal to 8 times), the cervix is not changed progressively, and the cervical tolerance is less than 80%.
3) Premature women meet diagnostic criteria: delivery is carried out in 28-37 weeks of gestation.
4) Women with term labor meet diagnostic criteria: pregnancy is greater than or equal to 37 weeks and <42 weeks.
5) All participants agreed to sign informed consent.
The exclusion criteria were as follows:
1) Does not correspond to the week of pregnancy.
2) Serious medical complications (heart disease, diabetes, etc.).
3) Pregnancy complications (hypertension, twins and multiple pregnancy, premature placenta peeling, amniotic fluid embolism, etc.).
4) Vaginal drugs, antibiotics or hormonal drugs were used in the first 3 months.
The collected samples were divided into 29 cases (34.52%) of full term labor (FB), 30 cases (35.71%) of pre-term labor (TPB), and 25 cases (29.76%) of premature labor (PB). The vagina was opened with a vaginal speculum and the post-vaginal vault secretion was removed with a sterile cotton swab. Immediately after collecting the material, the sterile cotton swab is placed in a protective solution (a fecal microorganism genome protective solution kit for medical treatment using a south core), and 16SrRNA is stored at 4-8 ℃.
The study was approved by the eighth institutional ethics committee of the university of chinese.
1.3DNA extraction, PCR amplification, pyrosequencing
Flushing the cotton swab in the protective liquid. Then, 1mL of the protective solution containing the sample was centrifuged at 12000rpm for 15min, and the supernatant was discarded. The vaginal secretion flora DNA was extracted strictly according to the "microscale flora DNA extraction kit" (LS-R-N-007H-50/100, longsee) protocol. The 16S rRNA gene was PCR amplified using Q5 high fidelity DNA polymerase using forward primers (3417F 5'-ACTCCTACGGGAGGCAGCA-3', SEQ ID NO: 1) and reverse primers (806R 5'-GGACTACHVGGGTWTCTAAT-3', SEQ ID NO: 2), wherein H represents A/C/T, V represents A/C/G, and W represents A/T. The PCR product was then detected by 2% agarose gel electrophoresis, and the target fragment was gel recovered using AxyPrep DNA gel recovery kit (AP-GX-50G, escherin). For sequencing the 16S rRNA gene amplicon, truSeq Nano DNA LT Library Prep Kit (Illumina Inc, calif., USA) was used to prepare an amplicon library. The sequencing reactions were performed using the Illumina MiSeq platform and the data were analyzed by the microbiological ecology quantitative research platform (QIIME, www.qiime.org) using default parameters.
Before assembly, sequence reads (reads) were first filtered using trimmatic v.0.32 software to remove low quality or ambiguous reads, including reads that do not exactly match the primer, sequences with overlap mismatch ratios higher than 0.05, and original reads shorter than 100 bp. When at least 10 reads overlap reads generated at opposite ends of the same DNA fragment, according to the overlapping relation between paired-end reads, the maximum allowable error rate of the overlapping area is 0.2, and FLASH is used for merging paired-end clean reads (data to be analyzed), and the spliced sequence is called raw read (original tag).
High quality sequences with a distance similarity of 97% or more are grouped into operational classification units (operational taxonomic units, OTUs) using a search algorithm. A representative sequence is then extracted from each operation class unit. Next, the chimeric sequence is detected and removed. Subsequently, the microbial diversity was analyzed using a variety of software and algorithms.
1.4 statistical analysis
p <0.05 is statistically significant for differences between groups. Statistical analysis was performed using GraphPad Prism 8.0 software using unpaired two-tailed t-test (Mann-Whitney U test). Principal Component Analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed on each pair of combinations of all samples. Linear Discriminant Analysis (LDA) effector (LEfSe) tubing and metadata are used to identify different microorganisms that distinguish pregnant women from term delivery.
2. Results
2.1 subject clinical data
The study included 29 term birth (FB) groups (34.52%), 30 pre-term labor (TPB) groups (35.71%), 25 pre-term labor (PB) groups (29.76%), and a summary of the three clinical data is shown in table 1.
TABLE 1 clinical data (x+ -sd) for term delivery group, pre-term delivery group and pre-term delivery group
2.2 vaginal flora diversity analysis
Taxonomic classification diversity analysis showed that the detected vaginal bacteria were largely classified into Firmicutes, actinomycetes (actionbacteria) and Proteobacteria (Proteobacteria) 3 (fig. 1A).
At the genus level, more bacteria were detected in the FB group and less bacteria were detected in the TPB group. Bifidobacteria (bifidobacteria) are relatively large in the FB group, pallidum (aerobacteria) are relatively large in the PB group, and Gardnerella (Gardnerella) and mirabilium (atobium) are relatively large in the TPB group (fig. 1B). Overall, the results show that most pregnant women have a vaginal ecosystem with a predominantly lactic acid flora. Gardnerella (Gardnerella) is enriched in TPB pregnant women and is widely reported as a pathogen that increases the risk of premature abortion in pregnant women.
Vaginal flora diversity was assessed at the unit level in groups FB, TPB and PB (figure 2). FB group average OTUs 23, TPB group average OTUs 20, PB group average OTUs 42. Of the three groups, the PB group OTUs was the highest (p < 0.01) and the TPB group was the lowest (p < 0.01). In vaginal flora diversity evaluation, the Chao1 index analysis showed that the Chao1 value of TPB group was also the lowest of the three groups (p < 0.01), consistent with the taxonomic classification results (FIG. 1B). There was no statistical significance for the differences in Shannon and Simpson values between the three groups (fig. 4-5).
While PCoA analysis cannot distinguish most pregnant women from three groups (FIG. 6), PLS-DA analysis can distinguish FB group from PB and TPB groups (FIG. 7). The vaginal flora structure of premature pregnant women is indicated to be different from that of full-term pregnant women.
2.3 differential flora analysis
The LEfSe algorithm was used to identify the differential distribution of microbiota between FB, TPB and PB groups. Fig. 8 to 9 show that S-mers and S-melaninogenica were found to have a differential distribution in FB and PB by LEfSe analysis (LDA Score (log 10) =4) (fig. 8 to 9). FIGS. 10 to 11 show that there was a difference in distribution between PB and TPB by LEfSe analysis of f-Leptotrichiaceae, g-Sneathia and p-Proteobacteria (LDA Score (log 10) =4) (FIGS. 10 to 11). Transfer analysis found that there were differences between the three groups for two (Prevotella melaninogenica and Serratia marcescens) and four species (stenotophomonas, caulobacter, sphingomonas and aseptic ca. S) (figures 12-17). The relative abundance of melaninogenica was highest in FB group, and the relative abundance of Serratia marcescens, stenotrophomonas, caulobacter, sphingomonas and aseptic ca ul was highest in PB group.
3. Discussion of the invention
Pre-term or premature delivery can severely affect the health of pregnant women and infants. Fortunately, pre-term labor can prolong the gestational weeks through early intervention, improving the pregnancy outcome. However, at present, no early diagnosis means for the pre-term labor exists, and the difficulty of diagnosing and timely treating the pre-term labor dangerous pregnant women is increased. Meanwhile, the mechanism behind premature labor is not clear, and one proven mechanism is associated with amniotic fluid microbial infection. Bacterial Vaginosis (BV) is associated with pro-inflammatory germ cytokines as a risk factor for premature birth. The uterine cavity and vagina are physiologically adjacent passages, and the effects of female genital tract microbial translocation interfering with the vaginal flora on the intrauterine flora have been demonstrated.
The research detects the vaginal flora structures of full-term, premature and pre-term pregnant women through 16S rRNA sequencing, explores the unique flora structures of pregnant women with different health conditions, searches for pre-term and premature potential early diagnosis markers, guides the use of clinical antibiotics, and improves the expression of H to be A/C/T;
v represents A/C/G; w represents A/T.
Our study has increased new evidence by analyzing vaginal microbiota of pregnant women of different health states, indicating that pregnant women with pre-term labor, premature labor and term delivery have unique vaginal microbiota structures. Healthy vaginal microecology is produced by hydrogen peroxide (H) 2 O 2 ) Is maintained by lactobacillus. Even kostin suggests that the risk of premature birth in women with normal vaginal microbiota is reduced by 75% and that the absence of lactic acid bacteria is the strongest risk factor compared to BV. Our results show that the lactic acid bacteria-based microbiota is present in most pregnant women. Vaginal flora diversity (OTUs index and Chao1 index) was lowest among the three groups in the TPB group (p<0.05). In addition, the relative abundance of p.melaninogenica is highest in FB group, and the relative abundance of s.marcescens, stenotrophomonas, caulobacter, sphingomonas and statical caseis is highest in PB group, and can be used as a potential marker for diagnosing health status of pregnant women.
In conclusion, the present study suggests different bacteria for pre-term delivery, premature delivery and term pregnant women, providing biomarkers for early diagnosis, early intervention, and improvement of pregnancy outcome for pre-term delivery and risk of premature delivery.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (3)
1. The application of a reagent for detecting the abundance of vaginal microorganisms in preparing auxiliary diagnostic kits for premature delivery and prematurity is characterized in that the vaginal microorganisms are one or more of Prevotella melanogenesis (Prevotella melaninogenica), serratia viscosa (Serratia marcescens), stenotrophomonas (stenotrophomonas), lactobacillus (Caulobacter), sphingomonas and non-adhesive sessile (Acca).
2. The use according to claim 1, wherein the reagent for detecting the abundance of a vaginal microorganism is a primer or probe for detecting a vaginal microorganism.
3. The use according to claim 1, wherein the reagent for detecting the abundance of vaginal microorganisms is a primer for detecting Prevotella melanogenus (Prevolvulanagenic), serratia viscosa (Serratia marcescens), stenotrophomonas (stentrophomonas), stemona (Caulobacter), sphingomonas (Sphingomonas), and Achromobacter non-sticking (Achromobacter) 16SrRNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310704577.XA CN116515992A (en) | 2023-06-14 | 2023-06-14 | Application of reagent for detecting vaginal microorganism abundance in preparation of premature labor and prematurity auxiliary diagnosis kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310704577.XA CN116515992A (en) | 2023-06-14 | 2023-06-14 | Application of reagent for detecting vaginal microorganism abundance in preparation of premature labor and prematurity auxiliary diagnosis kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116515992A true CN116515992A (en) | 2023-08-01 |
Family
ID=87399619
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310704577.XA Pending CN116515992A (en) | 2023-06-14 | 2023-06-14 | Application of reagent for detecting vaginal microorganism abundance in preparation of premature labor and prematurity auxiliary diagnosis kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116515992A (en) |
-
2023
- 2023-06-14 CN CN202310704577.XA patent/CN116515992A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mändar et al. | Complementary seminovaginal microbiome in couples | |
| CN110423804B (en) | Biomarker set for screening remaining abortion risk and screening method | |
| Ichiyama et al. | Analysis of vaginal and endometrial microbiota communities in infertile women with a history of repeated implantation failure | |
| CN107635571B (en) | Composition for treating pregnancy-related diseases containing extracellular vesicles derived from bacteria belonging to the genus Bacillus | |
| Cregger et al. | Reproductive microbiomes: using the microbiome as a novel diagnostic tool for endometriosis | |
| Lawton et al. | High prevalence of Mycoplasma genitalium in women presenting for termination of pregnancy | |
| CN113186311B (en) | Application of vaginal microorganism in differential diagnosis of chronic pelvic pain syndrome | |
| Li et al. | The effects of perineal disinfection on infant’s oral microflora after transvaginal examination during delivery | |
| Anglim et al. | The effect of local estrogen therapy on the urinary microbiome composition of postmenopausal women with and without recurrent urinary tract infections | |
| Huang et al. | Effects of group B streptococcus infection on vaginal micro-ecology and pregnancy outcomes of pregnant women in late pregnancy | |
| Zou et al. | The endometrial microbiota profile influenced pregnancy outcomes in patients with repeated implantation failure: A retrospective study | |
| CN116515992A (en) | Application of reagent for detecting vaginal microorganism abundance in preparation of premature labor and prematurity auxiliary diagnosis kit | |
| Franco-Acosta et al. | Emergence of genital infections due to Haemophilus pittmaniae and Haemophilus sputorum | |
| Hummel et al. | Evidence for the amnion-fetal gut-microbial axis in late gestation beef calves | |
| Frąszczak et al. | The analysis of Lactobacillus spp. distribution in the vaginal microbiota of Polish women with abnormal Pap smear result | |
| KR101522398B1 (en) | Composition for diagnosing human papillomavirus infection and cervical cancer | |
| Xiao et al. | Effects of emergency/nonemergency cervical cerclage on the vaginal microbiome of pregnant women with cervical incompetence | |
| Vladislavovna et al. | Microbiome of the uterus in women with unsuccessful in vitro fertilization attempts | |
| Gao et al. | Reproductive tract microbiota of women in childbearing age shifts upon type of gynecological infections and phase in physiological cycle | |
| Al-Muk et al. | Isolation of Gardnerella vaginalis from pregnant women with bacterial vaginosis in Basrah, Iraq | |
| Tong et al. | Longitudinal Study of Vaginal Microbiota in Pregnant Women Following in Vitro Fertilization | |
| Anukam et al. | Association between absence of vaginal lactobacilli PCR products and Nugent scores interpreted as bacterial vaginosis | |
| CN106701915A (en) | Preparation method and application of LH-PCR bacterial gene fingerprint spectrum for bacterial vaginosis | |
| WO2023085397A1 (en) | Detection method for hunner-type interstitial cystitis diagnosis marker, hunner-type interstitial cystitis diagnosis kit, and hunner-type interstitial cystitis therapeutic agent | |
| Wang et al. | Pelvic abscess associated with Actinomyces species‒a rare post-cesarean complication |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |