WO2022160359A1 - Application of intestinal microorganism as premature infant brain injury marker - Google Patents
Application of intestinal microorganism as premature infant brain injury marker Download PDFInfo
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- bipi
- enterobacteriaceae
- enterococcus
- staphylococcus
- brain injury
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Definitions
- the invention belongs to biotechnology, and specifically relates to the application of intestinal microorganisms as markers of brain damage in premature infants.
- BIPI Brain injury in premature infants
- BIPI can be divided into: (1) intracranial hemorrhage, including parenchymal hemorrhage and periventricular-intraventricular hemorrhage (PVH-IVH); cerebellum and brainstem injury; (2) white matter lesions: including cystic and non-cystic Periventricular leukomalacia (with focal necrosis) leukomalacia, PVL); non-necrotizing diffuse white matter damage, etc.; mainly PVH-IVH and PVL.
- 16SrRNA is a component of the 30S subunit in the prokaryotic ribosome
- 16SrDNA is the DNA sequence corresponding to the ribosomal 16SrRNA molecule in the genome.
- the gene is about 1540bp in length and exists in all bacterial chromosomal genomes. It contains 10 conserved regions and 9 variable regions. The conserved regions reflect the relationship between species, and the variable regions can reflect the differences between species, and the variation The degree is closely related to the development of the flora and is the most suitable indicator for identifying the phylogeny and classification of bacteria.
- the present invention uses 16S rDNA sequencing technology to dynamically sequence the fecal flora of premature infants, explores the colonization pattern of intestinal flora in the early life of premature infants and the changes of intestinal flora in the occurrence and development of BIPI, and provides early prevention and treatment of clinical use of probiotics.
- BIPI provides a theoretical basis.
- the present invention adopts the following technical scheme: the application of intestinal microorganisms as markers of brain injury in premature infants; the intestinal microorganisms are one of Enterobacteriaceae, Enterococcus and Staphylococcus or several.
- the invention discloses the application of intestinal microorganisms in the preparation of premature infant brain injury markers; the intestinal microorganisms are one of Enterobacteriaceae, Enterococcus, Staphylococcus or several.
- the invention discloses the application of a reagent for detecting the abundance of intestinal microbes in the preparation of premature infant brain injury markers; the intestinal microbes are Enterobacteriaceae, Enterococcus and Staphylococcus one or more of them.
- Reagents for detecting the abundance of gut microbes include primers or probes for detecting gut microbes; for example, primers for detecting Enterobacteriaceae, Enterococcus, and Staphylococcus 16SrRNA.
- the invention discloses a system for assisting diagnosis of brain injury in premature infants by using the intestinal microbes of the invention, comprising a sequencing module for sequencing the isolated intestinal flora nucleic acid samples (fecal DNA) to obtain sequencing results; According to the sequencing result, the relative abundance of the microbial markers in the intestinal flora is detected to obtain the relative abundance value; the comparison module compares the obtained relative abundance value of the microbial marker with the set value.
- the set value was the relative abundance of microbial markers obtained from the normal group (non-BIPI group).
- the intestinal microorganisms are composed of Enterobacteriaceae, Enterococcus and Staphylococcus.
- the brain injury of the premature infant is the brain injury of the premature very low birth weight infant.
- the present invention uses high-throughput sequencing technology to sequence the 16SrDNA gene of fecal samples of premature infants, and explores the influence of intestinal flora on the occurrence and development of BIPI in premature infants.
- the application of gut microbes composed of Staphylococcus as a marker of brain injury in premature infants provides a reliable basis for the prevention and treatment of BIPI.
- Figure 1 shows the dilution curve of the sample.
- Figure 2 is an OTU heat map of the relative abundance of Top50 on day 4.
- Figure 3 is an OTU heat map of the relative abundance of Top50 on day 28.
- Figure 4 shows the relative abundance difference of Enterobacteriaceae between the two groups.
- Figure 5 shows the relative abundance difference of Enterococcus between the two groups.
- Figure 6 shows the relative abundance difference of Staphylococcus between the two groups, respectively.
- Figure 7 shows the relative abundance difference of Klebsiella between the two groups.
- Figure 8 shows the relative abundance difference of Bacteroides between the two groups.
- Figure 9 shows the relative abundance difference of Pseudomonas between the two groups.
- Figure 10 is a box plot of the Chao1 index between BIPI and non-BIPI groups.
- Figure 11 is a box plot of the observed species index between BIPI and non-BIPI groups.
- Figure 12 is a box plot of the simpson index between the BIPI group and the non-BIPI group.
- Figure 13 is a box plot of the shannon index index between the BIPI group and the non-BIPI group.
- Figure 14 shows the PCA and PCoA conditions of the two groups at different times.
- marker refers to the risk level or diagnosis of brain injury due to the significant difference between the two groups when comparing brain-damaged premature infants with non-brain-damaged premature infants Substances that are the benchmark for disease occurrence.
- primer refers to an oligonucleotide, which may be single-stranded or double-stranded, long enough to initiate synthesis of the intended extension product in the presence of an inducer.
- the exact length of primers depends on many factors, including temperature, source of primers, and method of use.
- Enterobacteriaceae Enterobacteriaceae
- Enterococcus Enterococcus
- Staphylococcus Staphylococcus
- Example: The present invention discloses the application of Enterobacteriaceae, Enterococcus and Staphylococcus in the preparation of microbial markers for brain injury in premature infants; Enterobacteriaceae, Enterococcus Enterococcus and Staphylococcus are used as microbial markers of brain injury in premature infants.
- the present invention starts with the intestinal flora, with a 2018 From May 30th to September 30th, 2019, the premature infants who were admitted to the NICU of Soochow University Children's Hospital and/or the NICU of the Mother and Child Center of Suzhou Municipal Hospital within 24 hours after birth, birth weight ⁇ 1500g and hospitalization time ⁇ 28 days are:
- the application of markers provides a theoretical basis for the clinical application of probiotics for early prevention and early intervention of BIPI.
- Diagnostic criteria and grouping 2.2.1 Diagnostic criteria for brain injury in premature infants: According to the "Diagnostic Recommendations for Periventricular-Intraventricular Hemorrhage and Periventricular Leukomalacia in Premature Infants" and the 2012 edition of “Expert Consensus on Diagnosis and Prevention of Brain Injury in Premature Infants", combined with Neonatal behavioral neurological assessment (NBNA) was used for diagnosis. Imaging abnormalities and/or clinical manifestations, and/or NABA score ⁇ 35 were diagnosed as BIPI. Clinical manifestations of premature infants: apnea, depression, bradycardia, hypotension, hypertension or blood pressure fluctuations, changes in consciousness, convulsions, increased intracranial pressure, abnormal muscle tone, abnormal primitive reflexes, etc.
- PVH-IVH is divided into 4 grades: grade I bleeding is confined to the germinal stroma layer and does not enter the ventricle; grade II is intraventricular hemorrhage without ventriculomegaly; grade III is intraventricular hemorrhage and ventriculomegaly; grade IV is intraventricular hemorrhage Internal hemorrhage with periventricular infarction.
- PVL is divided into 4 grades according to the de Vries grading method: grade 1 is local strong echoes around the bilateral ventricles, ⁇ 7 days, and no cystic cavity after that; grade 2 is local strong echoes around the bilateral ventricles, which changes as early as 2 weeks after birth It is a local small cyst; grade III is a wide range of strong echoes around the ventricle, which transforms into extensive cysts as early as 2 weeks after birth, and some of the cysts can be fused into pieces; grade IV is a wide range of strong echoes around the ventricle, involving the subcortical superficial white. The periventricular and subcortical diffuse cystic changes were transformed as early as 2 weeks after birth.
- Non-hemorrhagic injury of white matter early white matter area shows high signal on T1WI, and low signal or isointensity on T2WI; later white matter area shows low signal or white matter volume reduction or signal on T1WI Disappeared, showed high signal on T2WI, and ventricular dilatation may occur in severe cases.
- 2Hemorrhagic injury In the early stage, it showed high signal on T1WI, low signal on T2WI, and high signal on T1WI and T2WI in the later stage.
- Diffusion-weighted magnetic resonance imaging (MRI-DWI) showed hyperintensity within 1 to 2 weeks, and later hypointense or isointense.
- the 20-item NBNA score is divided into 5 parts: 6 items of behavioral adaptability (ability to adapt to the external environment and stimuli), 4 items each of active muscle tone and passive muscle tone, and 3 items each of primitive reflex and general response. Scoring criteria: 0 points for failure to elicit and significant abnormality, 1 point for slight abnormality, 2 points for complete normality, and a full score of 40 points; all scores are evaluated based on the best behavioral performance, and an NBNA total score ⁇ 35 is considered abnormal.
- Antibiotic classification The classification of antibiotics is based on the relevant standards of the guidelines for the clinical application of antibiotics issued by the National Health and Family Planning Commission, the Jiangsuzhou Antimicrobial Clinical Application Classification Management Catalog and the Children's Hospital affiliated to Soochow University Children's Hospital.
- Unrestricted use antibacterial drugs that are safe, effective, less resistant and less expensive. Such as: Sulfonicillin sodium.
- Restricted use Compared with the unrestricted use of antibiotics, its clinical efficacy is good, but the price is higher, and there are limitations in safety and drug resistance. Such as: laoxetine, cefodizime, amoxicillin sulbactam, piperacillin sodium tazobactam and so on.
- Special use When using this type of antibiotics, it is necessary to doubly protect the bacteria to prevent the bacteria from developing resistance too quickly and lead to serious consequences. The adverse reactions are relatively obvious and should not be used arbitrarily; there are few clinical data studies on their efficacy or safety.
- VLBWI of preterm birth Clinical data of their mothers 1 General VLBWI status of preterm birth in the two groups: gender, gestational age, birth weight, mode of delivery, age at admission, whether it was in vitro fertilization, twins or multiples, small for gestational age, and Apgar score; 2 two groups General conditions of preterm VLBWI mothers: maternal age, gestational hypertension, gestational diabetes, amniotic fluid pollution, placental abruption, prenatal use of hormones, and antibiotic use; 3 The main primary underlying diseases and comorbidities of preterm VLBWI in the two groups: NRDS, BPD, frequent apnea, neonatal respiratory failure, NEC, sepsis, neonatal pneumonia, PNAC, PDA, ROP, EUGR, hemoglobulfen, and others.
- Specimen collection and preservation After the neonates were admitted to the hospital, the feces on the 1st, 4th, 7th, 14th, 21st, and 28th days after birth were collected. When sampling, the sampler needs to wear sterile gloves, use a sterile cotton swab to pick up fresh feces, avoid mixing feces and urine during collection, pay attention to aseptic operation, and use 3 1.5mL sterile non-enzyme EP tubes. Store the collected specimens in a -80°C refrigerator within ⁇ 2 hours.
- Cranial B-ultrasound examination B-ultrasound doctor performs bedside cranial ultrasound examination on premature infants, using Japanese TOSHIBA TA700 ultrasonic diagnostic instrument, the probe frequency is 3MHZ. During the examination, the newborn was in a resting state, and the brain was fully exposed. After applying the couplant to the probe, rotate the brain to different degrees to observe the structure of the brain and the blood flow of the cerebral arteries. After the examination, the B-ultrasound doctor judged the brain ultrasound images under the premise of unknown clinical data. Cranial MRI examination: sedation 15-30 minutes before examination, intravenous injection of phenobarbital sodium injection 10mg/Kg.
- NBNA score was performed by a rehabilitation physician. The examination begins between feedings, while sleeping, and is completed within 10 minutes. The room temperature is 22 ⁇ 28°C, the environment is quiet, and the light is semi-dark.
- the 72 matched groups were regarded as the non-BIPI group, with a total of 118 cases in the two groups, 58 males (49.2%) and 50.8% females, gestational age 27-32+2 weeks, average gestational age (30.17 ⁇ 1.28) weeks, The birth weight was 800 ⁇ 1500g, and the average birth weight was (1247.6 ⁇ 199.7)g. There was no significant difference in the general data and birth status of premature infants between the two groups (P>0.05), as shown in Table 1.
- Table 2 shows a comparison of the general conditions of preterm VLBWI mothers in the two groups during pregnancy.
- # is the chi-square test
- ⁇ is the continuity-corrected chi-square test
- — is not estimated.
- ⁇ is the Mann Whitney U test
- ⁇ is the t test
- # is the chi-square test
- ⁇ is the continuity-corrected chi-square test.
- Specimen detection Fecal DNA extraction (according to the instructions attached to the kit): (1) At room temperature, add 750 ⁇ L of lysis solution to the lysis tube, and use a sterile spoon to pick up 150 mg of the fecal sample and add it to the lysis tube. The frequency oscillator oscillated for 3 min. (2) Put the lysis tube into the centrifuge, centrifuge for 1 minute, add 400 ⁇ L of the obtained supernatant to No. 3 column F, cover No. 3 column F in a collection tube, and after centrifugation for 1 minute, discard the filter column. (3) Add 1200 ⁇ L of genomic DNA lysis solution to the collection tube obtained in the previous step and mix well by pipetting with a pipette. (4) Put the No.
- DNA concentration detection (1) Prepare a 0.5mL EP tube. (2) Label the tube cap. (3) Add 199 ⁇ L of the working solution to each EP tube, add 1 ⁇ L from each DNA liquid sample after extraction to the test tube containing the working solution, and mix by vortexing for 2 to 3 seconds. (4) Incubate all prepared samples for 2 minutes at room temperature. (5) Place the incubated samples in QubitTM3.0 DNA concentration was determined on a Fluorometer and the value recorded. (6) After the measurement is completed, the successfully extracted DNA solution (concentration ⁇ 5ng/ul) is temporarily stored in a refrigerator at -20°C for future use.
- MetaVx Library Construction Library Construction Amplifies Prokaryotic 16Sr Using PCR Primers Designed by Goldwisdom
- the two hypervariable regions of V3 ⁇ V4 on the RNA gene the specific steps are as follows: (1) 1st PCR Dilute the DNA extracted from the sample to 5ng/ ⁇ L as a template, and use the Primer and PCR enzymes in the existing kit to perform V3
- the ⁇ V4 region was amplified and experiments were performed concurrently using H2O as a negative control.
- prepare amplification premix Mix according to the following components according to the number of reactions + ⁇ , and dispense the prepared premix Mix into PCR reaction tubes.
- PCR product 25 ⁇ L Agencourt AMPure XP 45 ⁇ L.
- Mix well incubate for 5 min at room temperature, centrifuge briefly, stand on a magnetic stand for 3 min, and remove the supernatant.
- the first washing The reaction tube was placed on the magnetic stand still, 200 ⁇ l of freshly prepared 70% ethanol was added, and the reaction tube was allowed to stand at room temperature for 1 min to remove the 80% ethanol.
- the second washing Repeat the above 4) first washing operation, and centrifuge gently to remove the residual 70% ethanol. Drying of magnetic beads Dry at room temperature for 10 min.
- 2nd PCR Using the 1st PCR product obtained by the operation and purification as the template, the 2nd PCR is carried out and introduced into the sequencing adapter, and different combinations of Index can be added to the test sample primer. Prepare the following reactions (operated in Zone 1) on ice. In addition to the 1st PCR purified product, prepare the amplification premix Mix according to the following components according to the number of reactions + ⁇ , and dispense the prepared premix Mix into PCR reaction tubes.
- DNA sequencing (1) Library pretreatment: 1) Prepare denaturation reagents: Take 200 ⁇ L of 1N library denaturing solution for concentrated storage, add 800 ⁇ L of enzyme-free water to prepare 0.2N library denaturing solution, vortex to mix, centrifuge briefly, and set aside at room temperature for later use. 2) Denaturation of library: Take 5 ⁇ L of library (4nM) to the bottom of the low adsorption tube, add 5 ⁇ L of 0.2N library denaturation solution, and mix by pipetting. Close the lid and incubate at room temperature for 5 min, then place on ice immediately.
- the dilution curve can reflect species abundance and species uniformity.
- the length on the horizontal axis of the curve reflects species abundance. The larger the range, the higher the species abundance.
- the shape reflects the evenness of the species, and the flatter the curve, the higher the evenness of the species.
- Operational taxonomy unit is a taxa that is assumed in biological analysis. A sequence with a similarity higher than 97% in the sample sequence was defined as an OTU, and each OUT corresponds to a different 16S rDNA gene sequence, that is, each OTU corresponds to a different species. For the valid sequences of all samples, OTU clustering was performed with 97% consistency, and species annotation and species clustering were performed on the OTU sequences. In order to quickly and intuitively display the species composition and abundance information in the samples, the species annotation Heatmap corresponding to the OTU was constructed to display the OTU abundance information of the two groups of premature infants at different ages.
- the non-BIPI group was dominated by Enterobacteriaceae ( Enterobacteriaceae is the most abundant, followed by Enterococcus, Staphylococcus, and Streptococcus; at the genus level, the top five relative abundances of the two groups are Klebsiella, Enterobacter Genus (Enterobacteriaceae), Enterococcus (Enterococcus), Staphylococcus (Staphylococcus), Bacteroides (Bacteroides), including Enterobacteriaceae (Enterobacteriaceae), Enterococcus (Enterococcus), Staphylococcus (Staphylococcus) The relative abundance differences between the two groups are shown in Figure 4, Figure 5, and Figure 6.
- Alpha diversity refers to the diversity within a specific environment or ecosystem, through the use of species cumulative box plots, species diversity curves and a series of statistical analysis indices to evaluate the microorganisms in each sample Differences in community species richness and diversity.
- Chao1 index It is used to estimate the number of OTUs contained in the sample. The larger the value, the more species, and it is very sensitive to rare species.
- observed species index bioinformatics analysis in OTU units, observed Both species and Chao1 indices are used to represent the number of OTUs in the sample, indicating species richness.
- Shannon index It is mainly used to describe the disorder and uncertainty of OTU. The larger the Shannon index value, the more uniform the individual distribution.
- Simpson index used in ecology to quantitatively describe the biodiversity of a region. The larger the Simpson index value, the higher the community diversity.
- the chao1 index and observed species index of the BIPI group were higher than those of the non-BIPI group on the 4th to 28th days after birth, and were significantly higher than those of the non-BIPI group on the 21st and 28th days after birth (P ⁇ 0.05), suggesting that the intestinal flora of the BIPI group
- the richness was significantly higher than that of the non-BIPI group on the 21st and 28th days, as shown in Figures 10 and 11; the BIPI group's shannon index and simpson index were higher than those of the non-BIPI group on the 4th to 28th days after birth, and the 21st and 28th days after birth in the BIPI group days were significantly higher than those in the non-BIPI group (P ⁇ 0.05), suggesting that the BIPI group had a significantly increased diversity of intestinal flora on days 21 and 28 compared with the non-BIPI group, as shown in Figures 12 and 13.
- the abscissa is the sample collection time, and the ordinate is each index.
- the upper and lower ends of the box are the 75th percentile and the 25th percentile, respectively.
- the horizontal line in the middle of the box is the median, and the ends of the upper and lower tentacles are the 95th percentile.
- Percentile and 5th percentile dots are values outside the 95th and 5th percentile. * indicates P value ⁇ 0.05.
- Beta diversity analysis is an important part of biodiversity, and it is used to study the differences in species community structure between different samples or treatment groups. It is different from Alpha diversity, which compares the diversity differences between different samples (ecosystems), while Alpha diversity is mainly used to describe the species diversity of a single sample.
- Principal Component Analysis (PCA, Principal Component Analysis) is a method of applying variance decomposition to reduce the dimension of multidimensional data and extract the most important elements and structures in the data [51].
- Principal Coordinates Analysis Principal Coordinates Analysis
- PCoA Principal Co-ordinates Analysis
- PCoA selects the principal coordinate combination with the largest contribution rate for mapping analysis.
- PCA and PCoA between the two groups showed that there was a statistically significant difference in the Beta diversity between the two groups on the 21st and 28th days (P ⁇ 0.05), which were clustered together within the same group, and the BIPI group and the non-BIPI group at 28 days after birth
- the internal aggregation between the two groups is more obvious, as shown in Figure 14, A, C, E are the PCA conditions of the two groups at 4, 21, and 28 days, and B, D, and F are the PCoA conditions of the two groups at 4, 21, and 28 days, with different shapes and colors. Corresponds to the corresponding group name at the bottom of the graph.
- the gut microbiota plays an important role in human health and disease development.
- the neonatal period is an important period for the colonization and development of the gut microbiota, and disturbances in the gut microbiota during this period increase the risk of developing certain diseases in the future.
- the colonization and development of intestinal flora can promote the maturation of intestinal barrier and immune function, and have a profound impact on future health. Therefore, it is of great significance to study the characteristics of gut microbiota in preterm VLBWI and to explore whether it is related to the occurrence of BIPI.
- this study adopted the second-generation high-throughput sequencing technology based on the V3-V4 region of bacterial 16S rDNA.
- the detection rate of specimens on the first day after birth was low, which may be because the intestinal flora colonization of preterm VLBWI on the first day after birth was very small, and the duration of this study was long, although the samples were stored at -80 °C The refrigerator may still be degraded, so the samples on the first day were rejected.
- Example 2 The system for assisted diagnosis of brain injury in premature infants by using gut microbes of the present invention includes a sequencing module to sequence the isolated gut flora nucleic acid samples (fecal DNA) to obtain sequencing results; the abundance calculation module, according to According to the sequencing result, the relative abundance of the microbial markers in the intestinal flora is detected to obtain the relative abundance value; the comparison module compares the obtained relative abundance value of the microbial marker with the set value.
- the set value was the relative abundance of microbial markers obtained from the normal group (non-BIPI group).
- the application method of the present invention is simple and does not require complicated data calculation.
- Stool samples were collected from 3 cases of premature infants who were clinically diagnosed with BIPI and had no other brain diseases on the 7th day and 3 cases were born on the 4th day (not Example 1), and the same DNA extraction, amplification, sequencing, OTU species annotation and clustering methods were used to obtain the relative abundances of Enterobacteriaceae, Enterococcus, and Staphylococcus.
- Stool samples were collected from 3 cases of premature infants who were clinically diagnosed with BIPI and had no other brain diseases on the 7th day and 3 cases were born on the 4th day (not Example 1), and the same DNA extraction, amplification, sequencing, OTU species annotation and clustering methods were used to obtain the relative abundances of Enterobacteriaceae, Enterococcus, and Staphylococcus.
- Stool samples were collected from 4 premature infants who were clinically diagnosed without BIPI on the 7th day of life but had other diseases (2 cases of retinopathy of prematurity, 2 cases of premature infant apnea), and the same DNA extraction, amplification and sequencing as in Example 1 were used respectively.
- OTU species annotation and clustering methods to obtain the relative abundance of Enterobacteriaceae, Enterococcus, and Staphylococcus. Compared with the non-BIPI group in Example 1, the results of all samples are different. small, insignificant.
- the present invention discloses an assistant based on detecting the abundance of Enterobacteriaceae, Enterococcus and Staphylococcus.
- kit components are as follows: primer pairs for the detection of Enterobacteriaceae, Enterococcus, Staphylococcus 16SrRNA and other conventional reagents, such as DNA extraction reagents, reaction buffer, triphosphate base deoxygenation Nucleotides, Taq-polymerase, etc.
- BIPI pathogenesis protein
- its high-risk factors include prenatal, intrapartum and postnatal pathological factors.
- Current studies suggest that gestational age, birth weight, premature rupture of membranes, chorionitis, and asphyxia are the main prenatal and intrapartum risk factors for BIPI in preterm infants.
- the invention discloses the application of Enterobacteriaceae, Enterococcus and Staphylococcus in preparing microbial markers of brain injury in premature infants.
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Abstract
Disclosed in the present invention are an application of an intestinal microorganism as a premature infant brain injury marker, a diagnostic system, and a test kit. The gut microorganism is one or more of Enterobacteriaceae, Enterococcus, and Staphylococcus.
Description
本发明是属于生物技术，具体涉及肠道微生物作为早产儿脑损伤标志物的应用。The invention belongs to biotechnology, and specifically relates to the application of intestinal microorganisms as markers of brain damage in premature infants.
早产儿脑损伤（Brain injury in premature infants, BIPI）是指由于母亲存在产科合并症、围产期感染、脑缺血缺氧、生后呼吸衰竭、机械通气等多种因素造成早产儿出现脑缺血和/或出血性损伤，严重者可出现死亡或遗留多种神经系统后遗症。BIPI依据病理学改变可分为：①颅内出血，包括脑实质出血及脑室周围-脑室内出血（Periventricular-intraventricular hemorrhage, PVH-IVH）；小脑及脑干损伤；②白质病变：包括囊性和非囊性（伴局灶性坏死）脑室周围白质软化（Periventricular
leukomalacia, PVL）；非坏死性弥漫性白质损伤等；主要以PVH-IVH及PVL为主。人体内寄居的细菌与宿主之间存在着共生关系；肠神经元和肠免疫细胞具有相同的调节机制，可以协调大脑发育，且可对环境变化做出反应。目前尚未见关于肠道菌群与BIPI的相关研究报道。Brain injury in premature infants (BIPI) refers to cerebral insufficiency in premature infants caused by various factors such as maternal complications, perinatal infection, cerebral ischemia and hypoxia, postnatal respiratory failure, and mechanical ventilation. Blood and/or hemorrhagic injury, severe cases may lead to death or a variety of neurological sequelae. BIPI can be divided into: (1) intracranial hemorrhage, including parenchymal hemorrhage and periventricular-intraventricular hemorrhage (PVH-IVH); cerebellum and brainstem injury; (2) white matter lesions: including cystic and non-cystic Periventricular leukomalacia (with focal necrosis)
leukomalacia, PVL); non-necrotizing diffuse white matter damage, etc.; mainly PVH-IVH and PVL. There is a symbiotic relationship between the bacteria inhabiting the human body and the host; enteric neurons and intestinal immune cells share the same regulatory mechanisms that coordinate brain development and respond to environmental changes. So far, there are no related studies on gut microbiota and BIPI.
16SrRNA是原核生物核糖体中30S亚基的组成部分，16SrDNA是基因组中编码核糖体16SrRNA分子对应的DNA序列。该基因全长约1540bp，存在于所有细菌染色体基因组中，包含10个保守区和9个可变区，其中保守区反映了物种间的亲缘关系，可变区可体现物种间的差异，且变异程度与菌群发育密切相关，是最适于鉴别细菌系统发育和分类的指标。本发明通过16SrDNA测序技术对早产儿粪便菌群进行动态测序，探讨早产儿生命早期肠道菌群定植模式及肠道菌群在BIPI发生、发展过程中的变化，为临床上用益生菌早期防治BIPI提供理论依据。16SrRNA is a component of the 30S subunit in the prokaryotic ribosome, and 16SrDNA is the DNA sequence corresponding to the ribosomal 16SrRNA molecule in the genome. The gene is about 1540bp in length and exists in all bacterial chromosomal genomes. It contains 10 conserved regions and 9 variable regions. The conserved regions reflect the relationship between species, and the variable regions can reflect the differences between species, and the variation The degree is closely related to the development of the flora and is the most suitable indicator for identifying the phylogeny and classification of bacteria. The present invention uses 16S rDNA sequencing technology to dynamically sequence the fecal flora of premature infants, explores the colonization pattern of intestinal flora in the early life of premature infants and the changes of intestinal flora in the occurrence and development of BIPI, and provides early prevention and treatment of clinical use of probiotics. BIPI provides a theoretical basis.
本发明采用如下技术方案：肠道微生物作为早产儿脑损伤标志物的应用；所述肠道微生物为肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)中的一种或几种。The present invention adopts the following technical scheme: the application of intestinal microorganisms as markers of brain injury in premature infants; the intestinal microorganisms are one of Enterobacteriaceae, Enterococcus and Staphylococcus or several.
本发明公开了肠道微生物在制备早产儿脑损伤标志物中的应用；所述肠道微生物为肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)中的一种或几种。The invention discloses the application of intestinal microorganisms in the preparation of premature infant brain injury markers; the intestinal microorganisms are one of Enterobacteriaceae, Enterococcus, Staphylococcus or several.
本发明公开了检测肠道微生物丰度的试剂在制备早产儿脑损伤标志物中的应用；所述肠道微生物为肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)中的一种或几种。检测肠道微生物丰度的试剂包括检测肠道微生物的引物或者探针；比如检测检测肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)16SrRNA的引物。The invention discloses the application of a reagent for detecting the abundance of intestinal microbes in the preparation of premature infant brain injury markers; the intestinal microbes are Enterobacteriaceae, Enterococcus and Staphylococcus one or more of them. Reagents for detecting the abundance of gut microbes include primers or probes for detecting gut microbes; for example, primers for detecting Enterobacteriaceae, Enterococcus, and Staphylococcus 16SrRNA.
本发明公开了利用本发明肠道微生物辅助诊断早产儿脑损伤的系统，包括测序模块，对分离的肠道菌群核酸样本（粪便DNA）进行测序，以获得测序结果；丰度计算模块，根据测序结果，对肠道菌群中的所述微生物标志物的相对丰度进行检测，得到相对丰度值；比较模块，将得到的微生物标志物相对丰度值与设定值进行比较。设定值为正常组（非BIPI组）得到的微生物标志物相对丰度值。The invention discloses a system for assisting diagnosis of brain injury in premature infants by using the intestinal microbes of the invention, comprising a sequencing module for sequencing the isolated intestinal flora nucleic acid samples (fecal DNA) to obtain sequencing results; According to the sequencing result, the relative abundance of the microbial markers in the intestinal flora is detected to obtain the relative abundance value; the comparison module compares the obtained relative abundance value of the microbial marker with the set value. The set value was the relative abundance of microbial markers obtained from the normal group (non-BIPI group).
优选的，所述肠道微生物由肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)组成。Preferably, the intestinal microorganisms are composed of Enterobacteriaceae, Enterococcus and Staphylococcus.
优选的，早产儿脑损伤为早产极低出生体重儿脑损伤。Preferably, the brain injury of the premature infant is the brain injury of the premature very low birth weight infant.
目前尚未见关于肠道菌群与BIPI的相关研究报道，出生后肠道菌群的变化和神经系统的发育不成熟相结合，使早产儿成为研究初始肠道菌群定植对大脑发育影响的独特人群。因此，研究早产VLBWI肠道菌群定植情况及变化，对于研究BIPI有相当大的意义，未来有可能为儿童脑瘫及认知功能障碍的早期识别和早期干预提供依据。本发明通过高通量测序技术对早产儿粪便样本16SrDNA基因进行测序，探讨肠道菌群对早产儿BIPI发生、发展的影响，首次公开了由肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)组成的肠道微生物作为早产儿脑损伤标志物的应用，为BIPI的预防及治疗提供可靠依据。There have been no reports on the relationship between gut microbiota and BIPI. The combination of postnatal changes in gut microbiota and immaturity of the nervous system makes preterm infants unique for studying the effects of initial gut microbiota colonization on brain development. crowd. Therefore, studying the colonization and changes of intestinal flora in preterm VLBWI is of great significance for the study of BIPI, and may provide a basis for early identification and early intervention of children with cerebral palsy and cognitive dysfunction in the future. The present invention uses high-throughput sequencing technology to sequence the 16SrDNA gene of fecal samples of premature infants, and explores the influence of intestinal flora on the occurrence and development of BIPI in premature infants. The application of gut microbes composed of Staphylococcus as a marker of brain injury in premature infants provides a reliable basis for the prevention and treatment of BIPI.
图1为样本的稀释性曲线。Figure 1 shows the dilution curve of the sample.
图2为第4天Top50相对丰度的OTU热图。Figure 2 is an OTU heat map of the relative abundance of Top50 on day 4.
图3为第28天Top50相对丰度的OTU热图。Figure 3 is an OTU heat map of the relative abundance of Top50 on day 28.
图4分别是肠杆菌属（Enterobacteriaceae）在两组间的相对丰度差异。Figure 4 shows the relative abundance difference of Enterobacteriaceae between the two groups.
图5分别是肠球菌属(Enterococcus)在两组间的相对丰度差异。Figure 5 shows the relative abundance difference of Enterococcus between the two groups.
图6分别是葡萄球菌属(Staphylococcus)在两组间的相对丰度差异。Figure 6 shows the relative abundance difference of Staphylococcus between the two groups, respectively.
图7分别为克雷伯氏菌属(Klebsiella)在两组间的相对丰度差异。Figure 7 shows the relative abundance difference of Klebsiella between the two groups.
图8分别为拟杆菌属(Bacteroides)在两组间的相对丰度差异。Figure 8 shows the relative abundance difference of Bacteroides between the two groups.
图9分别为假单胞菌属 (Pseudomonas)在两组间的相对丰度差异。Figure 9 shows the relative abundance difference of Pseudomonas between the two groups.
图10为BIPI与非BIPI组chao1指数箱型图。Figure 10 is a box plot of the Chao1 index between BIPI and non-BIPI groups.
图11为BIPI与非BIPI组observed species指数箱型图。Figure 11 is a box plot of the observed species index between BIPI and non-BIPI groups.
图12 为BIPI组与非BIPI组simpson指数箱型图。Figure 12 is a box plot of the simpson index between the BIPI group and the non-BIPI group.
图13为BIPI组与非BIPI组shannon指数指数箱型图。Figure 13 is a box plot of the shannon index index between the BIPI group and the non-BIPI group.
图14为不同时间两组的PCA和PCoA情况。Figure 14 shows the PCA and PCoA conditions of the two groups at different times.
本发明中，“标志物”是指，将脑损伤的早产儿与非脑损伤的早产儿进行比较时，由于在两个组别中存在有明显差别而可以成为脑损伤的的危险程度或者诊断是否发生疾病时的基准的物质。In the present invention, "marker" refers to the risk level or diagnosis of brain injury due to the significant difference between the two groups when comparing brain-damaged premature infants with non-brain-damaged premature infants Substances that are the benchmark for disease occurrence.
在本发明中，“引物”表示寡核苷酸，可以是单链或双链，足够长以在诱导剂存在下引发合成预期的延伸产物。引物的确切长度依赖于很多因素，其中包括温度、引物来源和使用方法。例如，通常含有15-25个或更多核苷酸，根据本发明公开的肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)，对于本领域技术人员容易根据常规方法知道对于肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)16SrRNA的引物。In the present invention, "primer" refers to an oligonucleotide, which may be single-stranded or double-stranded, long enough to initiate synthesis of the intended extension product in the presence of an inducer. The exact length of primers depends on many factors, including temperature, source of primers, and method of use. For example, usually containing 15-25 or more nucleotides, according to the disclosure of the present invention Enterobacteriaceae (Enterobacteriaceae), Enterococcus (Enterococcus), Staphylococcus (Staphylococcus), for those skilled in the art easily according to conventional methods Primers for Enterobacteriaceae, Enterococcus, and Staphylococcus 16SrRNA are known.
统计学方法：数据统计分析使用SPSS22.0软件进行。正态分布的计量资料以均数±标准差（中位数±标准差，Mean±SD）表示，经方差齐性（Levene检验）检验后，两组间比较用独立样本t检验；非正态分布计量资料以中位数和第25、75百分位数[M (P25, P75)]表示，两组间采用Mann Whitney U检验，计数资料中采用卡方检验或连续性校正卡方检验（n≥40，1≤Tmin<5）分析，将两组间比较有差异的因素行Logistic回归分析，得出BIPI生后的高危因素，P<0.05认为差异具有统计学意义。Statistical methods: Statistical analysis of data was performed using SPSS 22.0 software. The normally distributed measurement data are expressed as mean±standard deviation (median±standard deviation, Mean±SD). After the homogeneity of variance (Levene test) test, the independent samples t test was used for comparison between two groups; non-normal The distribution measurement data were expressed as the median and the 25th and 75th percentiles [M (P25, P75)], the Mann Whitney U test was used between the two groups, and the chi-square test or the continuity-corrected chi-square test was used for the count data ( n≥40, 1≤Tmin<5) analysis, Logistic regression analysis was performed on the factors with differences between the two groups, and the high-risk factors of BIPI after birth were obtained. P<0.05 considered the difference to be statistically significant.
实施例：本发明公开了肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)在制备早产儿脑损伤微生物标志物中的应用；以肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)作为早产儿脑损伤微生物标志物。通过肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)的检测虽然不能直接获得诊断结果，但是可以作为中间信息，辅助早期识别发生BIPI的高危儿，早期干预，探讨生命早期的肠道菌群定植模式及肠道菌群在BIPI发生过程中的变化，为临床应用益生菌早期预防和早期干预BIPI提供理论依据。
Example: The present invention discloses the application of Enterobacteriaceae, Enterococcus and Staphylococcus in the preparation of microbial markers for brain injury in premature infants; Enterobacteriaceae, Enterococcus Enterococcus and Staphylococcus are used as microbial markers of brain injury in premature infants. Although the detection of Enterobacteriaceae, Enterococcus and Staphylococcus cannot directly obtain diagnostic results, it can be used as intermediate information to assist in early identification of high-risk children with BIPI, early intervention, and exploration of life The early colonization pattern of intestinal flora and the changes of intestinal flora during the occurrence of BIPI provide a theoretical basis for the clinical application of probiotics for early prevention and early intervention of BIPI.
为了验证以肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)作为早产儿脑损伤微生物标志物的准确性以及特异性，本发明从肠道菌群入手，以2018年05月30日至2019年09月30日于生后24小时之内入苏州大学附属儿童医院NICU和/或苏州市立医院本部母子中心NICU、出生体重<1500g且住院时间≥28天的早产儿为研究对象，通过16S rDNA测序对早产VLBWI粪便菌群进行动态测序，探讨生命早期的肠道菌群定植模式及肠道菌群在BIPI发生过程中的变化，验证了肠道微生物作为早产儿脑损伤标志物的应用，为临床应用益生菌早期预防和早期干预BIPI提供理论依据。In order to verify the accuracy and specificity of using Enterobacteriaceae, Enterococcus, and Staphylococcus as microbial markers for brain injury in premature infants, the present invention starts with the intestinal flora, with a 2018 From May 30th to September 30th, 2019, the premature infants who were admitted to the NICU of Soochow University Children's Hospital and/or the NICU of the Mother and Child Center of Suzhou Municipal Hospital within 24 hours after birth, birth weight <1500g and hospitalization time ≥28 days are: The research subjects, dynamic sequencing of preterm VLBWI fecal flora by 16S rDNA sequencing, to explore the intestinal flora colonization pattern in early life and the changes of intestinal flora during the occurrence of BIPI, and to verify the role of intestinal microorganisms as brain injury in premature infants. The application of markers provides a theoretical basis for the clinical application of probiotics for early prevention and early intervention of BIPI.
研究对象：2.1.1入选标准：以2018年05月30日至2019年09月30期间于生后24小时之内入苏州大学附属儿童医院NICU和/或苏州市立医院母子中心NICU、出生体重<1500 g且住院时间≥28天的早产儿为研究对象。本研究获患儿家属知情同意及苏州大学附属儿童医院/苏州市立医院伦理委员会批准。Research objects: 2.1.1 Inclusion criteria: from May 30, 2018 to September 30, 2019, they were admitted to the NICU of the Children's Hospital of Soochow University and/or the NICU of the Mother and Child Center of Suzhou Municipal Hospital within 24 hours after birth, birth weight < Preterm infants who were 1500 g and hospitalized for ≥28 days were included in the study. This study obtained the informed consent of the families of the children and was approved by the Ethics Committee of the Children's Hospital of Soochow University/Suzhou Municipal Hospital.
排除标准。（1）入院时龄>24小时，且住院时间<28（2）出生体重>1500g（3）先天性胃肠道及神经系统发育畸形者（4）染色体异常者（5）遗传代谢紊乱所致脑损伤及胆红素脑病、宫内TORCH感染及生后中枢神经系统感染者（6）生后28天内母乳喂养者（7）住院期间行外科手术者（8）入院积极抢救治疗数小时之内死亡者（9）临床资料不完善者。Exclusion criteria. (1) Age at admission > 24 hours, and hospitalization time < 28 (2) Birth weight > 1500g (3) Congenital malformations of gastrointestinal tract and nervous system development (4) Chromosomal abnormalities (5) Caused by genetic and metabolic disorders Brain injury, bilirubin encephalopathy, intrauterine TORCH infection and postnatal central nervous system infection (6) Breastfeeding within 28 days after birth (7) Surgery during hospitalization (8) Active rescue within a few hours after admission The deceased (9) those with incomplete clinical data.
诊断标准及分组：2.2.1早产儿脑损伤诊断标准：根据《早产儿脑室周围-脑室内出血与脑室周围白质软化的诊断建议》及2012版《早产儿脑损伤诊断与防治专家共识》，并结合新生儿行为神经测定（neonatal
behavioral neurological assessment, NBNA）进行诊断。有影像学异常和/或临床表现，和/或NABA评分<35分诊断为BIPI。早产儿临床表现：可有呼吸暂停、抑制状态、心动过缓、低血压、高血压或血压波动、意识改变、惊厥、颅内压增高、肌张力异常、原始反射异常等表现，也可不伴临床表现及体征。影像学诊断：因BIPI早产儿症状及体征不典型，明确病理改变需完善影像学检查。（1）头颅B超：PVH-IVH及PVL的诊断标准：
根据Papile分级法，PVH-IVH分为4级：Ⅰ级出血局限于生发基质层，未入脑室；Ⅱ级为脑室内出血，不伴脑室扩大；Ⅲ级为脑室内出血及脑室扩大；Ⅳ级为脑室内出血伴脑室周围梗死。
PVL根据de Vries分级法分为4级：Ⅰ级为双侧脑室周围局部强回声，≥7天，其后无囊腔；Ⅱ级为双侧脑室周围局部强回声，最早在生后2周转变为局部小囊腔；Ⅲ级为脑室周围广泛性强回声，最早在生后2周转变为广泛囊腔，部分囊腔可融合成片；Ⅳ级为脑室周围广泛强回声，累及皮质下浅表白质，最早在生后2周转变为脑室周围和皮质下弥漫性囊腔改变。（2）头颅MRI表现：①白质非出血性损伤：早期白质区域在T1WI表现为高信号，在T2WI表现为低信号或等信号；后期白质区域在T1WI上表现为低信号或白质容积减少或信号消失，在T2WI上表现为高信号，严重者可出现脑室扩张。②出血性损伤：早期在T1WI上表现为高信号，在T2WI上为低信号，后期在T1WI、T2WI上均为高信号。
弥散加权磁共振成像（MRI-DWI）在1~2周内表现为高信号，后期表现为低信号或等信号。20项NBNA评分分为5部分：行为适应能力6项（适应外界环境及刺激的能力），主动肌张力及被动肌张力各4项，原始反射及一般反应各3项。评分标准：未能引出和显著不正常者0分，轻微不正常1分，完全正常2分，满分40分；评分均以行为表现最优时评定，NBNA总分＜35分为异常。抗生素分级：抗生素的分级根据国家卫生计生委发布的抗菌药物临床应用指导原则、江苏省抗菌药物临床应用分级管理目录及苏州大学附属儿童医院抗菌药物临床应用分级管理目录的相关标准。（1）非限制使用：安全、有效、耐药性较小、价格较低的抗菌药物。如：磺苄西林钠。（2）限制使用：相较于非限制使用抗生素，其临床疗效好，但价格较高，安全性、耐药性存在局限性。如：拉氧头孢、头孢地嗪、阿莫西林舒巴坦、哌拉西林钠他唑巴坦等。（3）特殊使用：使用这类抗生素时需要倍加保护以免细菌过快产生耐药而导致严重后果，不良反应相对明显，不宜随意使用；关于其疗效或安全性的临床资料研究尚少，或并不优于现用药物者；新上市的抗菌药物；药品价格高。如：万古霉素、美罗培南、利奈唑胺等。研究分组：根据BIPI诊断标准，将发展为BIPI的早产VLBWI作为BIPI组，在未发生BIPI的早产VLBWI中随机抽取一般资料与BIPI组相匹配的早产儿作为非BIPI组。
Diagnostic criteria and grouping: 2.2.1 Diagnostic criteria for brain injury in premature infants: According to the "Diagnostic Recommendations for Periventricular-Intraventricular Hemorrhage and Periventricular Leukomalacia in Premature Infants" and the 2012 edition of "Expert Consensus on Diagnosis and Prevention of Brain Injury in Premature Infants", combined with Neonatal behavioral neurological assessment (NBNA) was used for diagnosis. Imaging abnormalities and/or clinical manifestations, and/or NABA score <35 were diagnosed as BIPI. Clinical manifestations of premature infants: apnea, depression, bradycardia, hypotension, hypertension or blood pressure fluctuations, changes in consciousness, convulsions, increased intracranial pressure, abnormal muscle tone, abnormal primitive reflexes, etc. manifestations and signs. Imaging diagnosis: Because the symptoms and signs of BIPI premature infants are not typical, imaging examinations should be improved to clarify the pathological changes. (1) Cranial B-ultrasound: Diagnostic criteria for PVH-IVH and PVL: According to the Papile grading method, PVH-IVH is divided into 4 grades: grade I bleeding is confined to the germinal stroma layer and does not enter the ventricle; grade II is intraventricular hemorrhage without ventriculomegaly; grade III is intraventricular hemorrhage and ventriculomegaly; grade IV is intraventricular hemorrhage Internal hemorrhage with periventricular infarction. PVL is divided into 4 grades according to the de Vries grading method: grade 1 is local strong echoes around the bilateral ventricles, ≥ 7 days, and no cystic cavity after that; grade 2 is local strong echoes around the bilateral ventricles, which changes as early as 2 weeks after birth It is a local small cyst; grade III is a wide range of strong echoes around the ventricle, which transforms into extensive cysts as early as 2 weeks after birth, and some of the cysts can be fused into pieces; grade IV is a wide range of strong echoes around the ventricle, involving the subcortical superficial white. The periventricular and subcortical diffuse cystic changes were transformed as early as 2 weeks after birth. (2) Cranial MRI manifestations: ① Non-hemorrhagic injury of white matter: early white matter area shows high signal on T1WI, and low signal or isointensity on T2WI; later white matter area shows low signal or white matter volume reduction or signal on T1WI Disappeared, showed high signal on T2WI, and ventricular dilatation may occur in severe cases. ②Hemorrhagic injury: In the early stage, it showed high signal on T1WI, low signal on T2WI, and high signal on T1WI and T2WI in the later stage. Diffusion-weighted magnetic resonance imaging (MRI-DWI) showed hyperintensity within 1 to 2 weeks, and later hypointense or isointense. The 20-item NBNA score is divided into 5 parts: 6 items of behavioral adaptability (ability to adapt to the external environment and stimuli), 4 items each of active muscle tone and passive muscle tone, and 3 items each of primitive reflex and general response. Scoring criteria: 0 points for failure to elicit and significant abnormality, 1 point for slight abnormality, 2 points for complete normality, and a full score of 40 points; all scores are evaluated based on the best behavioral performance, and an NBNA total score <35 is considered abnormal. Antibiotic classification: The classification of antibiotics is based on the relevant standards of the guidelines for the clinical application of antibiotics issued by the National Health and Family Planning Commission, the Jiangsu Province Antimicrobial Clinical Application Classification Management Catalog and the Children's Hospital Affiliated to Soochow University Children's Hospital. (1) Unrestricted use: antibacterial drugs that are safe, effective, less resistant and less expensive. Such as: Sulfonicillin sodium. (2) Restricted use: Compared with the unrestricted use of antibiotics, its clinical efficacy is good, but the price is higher, and there are limitations in safety and drug resistance. Such as: laoxetine, cefodizime, amoxicillin sulbactam, piperacillin sodium tazobactam and so on. (3) Special use: When using this type of antibiotics, it is necessary to doubly protect the bacteria to prevent the bacteria from developing resistance too quickly and lead to serious consequences. The adverse reactions are relatively obvious and should not be used arbitrarily; there are few clinical data studies on their efficacy or safety. No better than existing drugs; newly marketed antibacterial drugs; high drug prices. Such as: vancomycin, meropenem, linezolid, etc. Study grouping: According to the BIPI diagnostic criteria, the premature VLBWI with BIPI was selected as the BIPI group, and the preterm infants whose general information matched the BIPI group were randomly selected from the preterm VLBWI without BIPI as the non-BIPI group.
研究方法：前瞻性研究：采用前瞻性研究对符合入选标准的早产VLBWI进行观察研究，采集早产VLBWI的基本信息及母亲孕期基本资料，收集所有入选的早产VLBWI的粪便样本。抽取已采集好的BIPI组与非BIPI组的临床信息和粪便样本，对两组不同时间点的粪便标本采用16Sr
DNA高通量测序技术进行检测并进行生物信息学分析。具体如下：（1）分别于生后留取第1、4、7、14、21、28天的粪便标本；（2）所有患儿于生后第3天内完善头颅B超，有异常者每周复查一次，无异常者2周复查一次直至出院，于第28天完善头颅MRI检查；于生后26~28天行新生儿行为神经测定并行神经20项评分；（3）搜集并记录早产VLBWI及其母亲临床资料：①两组早产VLBWI一般情况：性别、胎龄、出生体重、生产方式、入院时龄、是否为试管婴儿、双胎或多胎、小于胎龄儿、Apgar评分；②两组早产VLBWI母亲一般情况：母亲年龄、妊娠期高血压、妊娠期糖尿病、羊水污染、胎盘早剥、产前是否使用激素、使用抗生素；③两组早产VLBWI主要原发基础疾病及合并症：NRDS、BPD、频发呼吸暂停、新生儿呼吸衰竭、NEC、败血症、新生儿肺炎、PNAC、PDA、ROP、EUGR、血红蛋白浓度<90g/L、血小板总数<100×109/L；④早产VLBWI住院期间的治疗措施：抗生素使用情况、氧疗时间、机械通气、机械通气时长、呼吸机模式（常频模式、高频模式）、输血≥3次、开奶日龄、肠外营养时间等。标本采集与保存：在新生儿入院后，留取其生后第1、4、7、14、21、28天的粪便。取样时，取样者需佩戴无菌手套，用无菌棉签掏取新鲜粪便，采集时注意避免粪尿混合，注意无菌操作，用3个1.5mL无菌无酶EP管分装，在30分钟～2小时内将采集的标本放置在-80℃冰箱保存。头颅B超检查：由B超科医生对早产儿实施床边颅脑超声检查，采用日本TOSHIBA
TA700型超声诊断仪，探头频率为3MHZ。检查时新生儿处于静息状态下，充分暴露颅脑，将耦合剂涂抹探头后，在脑部做不同程度旋转，观察颅脑结构、大脑动脉血流等情况。检查结束后由B超科医师在未知临床资料的前提下判断颅脑超声图像。头颅MRI检查：检查前15-30分钟镇静，静注苯巴比妥钠注射液10mg/Kg。采用美国GE
3.0T超导高场磁共振仪，64通头部线圈扫面，对早产儿轴位及矢状面自旋回波序列。检查结果由放射科医师在未知临床资料的前提下进行阅片和诊断。NBNA评分：由康复科医师进行NBNA评分。检查在两次喂奶中间、睡眠状态下开始，10分钟内完成。室温22~28℃，环境安静，光线半暗。Research methods: Prospective study: Prospective study was used to conduct an observational study on VLBWI of preterm birth who met the inclusion criteria. The basic information of VLBWI of preterm birth and the basic information of mother's pregnancy were collected, and the stool samples of all selected VLBWI of preterm birth were collected. The collected clinical information and stool samples of the BIPI group and the non-BIPI group were collected, and 16Sr was used for the stool samples of the two groups at different time points.
DNA high-throughput sequencing technology for detection and bioinformatics analysis. The details are as follows: (1) fecal specimens were collected on the 1st, 4th, 7th, 14th, 21st, and 28th days after birth; (2) B-ultrasound of the head of all children was completed within the 3rd day after birth. Re-examination once a week, no abnormality once every 2 weeks until discharge, complete head MRI examination on the 28th day; neonatal behavioral neurometry and 20-item neurological score on 26-28 days after birth; (3) Collect and record VLBWI of preterm birth Clinical data of their mothers: ① General VLBWI status of preterm birth in the two groups: gender, gestational age, birth weight, mode of delivery, age at admission, whether it was in vitro fertilization, twins or multiples, small for gestational age, and Apgar score; ② two groups General conditions of preterm VLBWI mothers: maternal age, gestational hypertension, gestational diabetes, amniotic fluid pollution, placental abruption, prenatal use of hormones, and antibiotic use; ③ The main primary underlying diseases and comorbidities of preterm VLBWI in the two groups: NRDS, BPD, frequent apnea, neonatal respiratory failure, NEC, sepsis, neonatal pneumonia, PNAC, PDA, ROP, EUGR, hemoglobin concentration <90g/L, total platelet count <100×109/L; ④VLBWI of preterm birth during hospitalization Treatment measures: antibiotic usage, time of oxygen therapy, mechanical ventilation, duration of mechanical ventilation, ventilator mode (normal frequency mode, high frequency mode), blood transfusion ≥3 times, age of starting milk, parenteral nutrition time, etc. Specimen collection and preservation: After the neonates were admitted to the hospital, the feces on the 1st, 4th, 7th, 14th, 21st, and 28th days after birth were collected. When sampling, the sampler needs to wear sterile gloves, use a sterile cotton swab to pick up fresh feces, avoid mixing feces and urine during collection, pay attention to aseptic operation, and use 3 1.5mL sterile non-enzyme EP tubes. Store the collected specimens in a -80°C refrigerator within ~2 hours. Cranial B-ultrasound examination: B-ultrasound doctor performs bedside cranial ultrasound examination on premature infants, using Japanese TOSHIBA
TA700 ultrasonic diagnostic instrument, the probe frequency is 3MHZ. During the examination, the newborn was in a resting state, and the brain was fully exposed. After applying the couplant to the probe, rotate the brain to different degrees to observe the structure of the brain and the blood flow of the cerebral arteries. After the examination, the B-ultrasound doctor judged the brain ultrasound images under the premise of unknown clinical data. Cranial MRI examination: sedation 15-30 minutes before examination, intravenous injection of phenobarbital sodium injection 10mg/Kg. US GE
3.0T superconducting high-field magnetic resonance apparatus, 64-channel head coil scanning, axial and sagittal spin echo sequences for premature infants. The examination results were interpreted and diagnosed by a radiologist under the premise of unknown clinical data. NBNA score: NBNA score was performed by a rehabilitation physician. The examination begins between feedings, while sleeping, and is completed within 10 minutes. The room temperature is 22~28℃, the environment is quiet, and the light is semi-dark.
表1 两组早产VLBWI一般情况比较（中位数±标准差）。Table 1 Comparison of the general conditions of VLBWI of preterm labor between the two groups (median ± standard deviation).
临床基本信息与资料：两组早产VLBWI一般情况比较：研究期间共观察189例早产VLBWI，排除入院未经抢救即死亡或积极抢救治疗数小时之内死亡22例，住院不满28天26例，外科手术8例，先天性胃壁缺如1例，中枢神经系统感染4例，染色体异常1例，其中发生脑损伤46例为BIPI组，未发生BIPI的81例早产VLBWI中，随机抽取一般资料与BIPI组相匹配的72例作为非BIPI组，两组共118例，男58例（49.2%），女（50.8%），胎龄27~32+2周，平均胎龄（30.17±1.28）周，出生体重800~1500g，平均出生体重（1247. 6±199.7）g。两组早产儿一般资料、出生情况比较均无明显差异（P>0.05），详见表 1。Basic clinical information and data: Comparison of the general conditions of VLBWI in two groups of preterm births: A total of 189 cases of preterm birth VLBWI were observed during the study period, excluding 22 cases of death without rescue or active rescue within a few hours after admission, 26 cases of hospitalization less than 28 days, surgical There were 8 cases of operation, 1 case of congenital absence of gastric wall, 4 cases of central nervous system infection, and 1 case of chromosomal abnormality. Among them, 46 cases of brain injury were in the BIPI group. Among the 81 cases of premature birth without BIPI, the general data and BIPI were randomly selected. The 72 matched groups were regarded as the non-BIPI group, with a total of 118 cases in the two groups, 58 males (49.2%) and 50.8% females, gestational age 27-32+2 weeks, average gestational age (30.17±1.28) weeks, The birth weight was 800~1500g, and the average birth weight was (1247.6±199.7)g. There was no significant difference in the general data and birth status of premature infants between the two groups (P>0.05), as shown in Table 1.
注：†为 t 检验，#为卡方检验。Note: † is t test, # is chi-square test.
3.1.2 两组早产VLBWI母亲孕期一般情况比较见表2。3.1.2 Table 2 shows a comparison of the general conditions of preterm VLBWI mothers in the two groups during pregnancy.
表2 两组早产VLBWI母亲孕期一般情况比较（例，%）。Table 2 Comparison of the general conditions of preterm VLBWI mothers in the two groups during pregnancy (cases, %).
注：#为卡方检验，※为连续性校正卡方检验，——为未能估计。Note: # is the chi-square test, ※ is the continuity-corrected chi-square test, and — is not estimated.
两组早产VLBWI原发基础疾病及合并症比较见表 3
[;两组早产VLBWI住院期间治疗情况比较见表 4。
The comparison of the primary underlying diseases and comorbidities of the two groups of preterm VLBWI is shown in Table 3 [; the comparison of the treatment conditions of the two groups of preterm VLBWI during hospitalization is shown in Table 4.
表3 两组早产VLBWI原发基础疾病和并发症发生情况比较（例，%）。Table 3 Comparison of the primary underlying diseases and complications of preterm VLBWI between the two groups (cases, %).
注：#为卡方检验。Note: # is the chi-square test.
表4 两组早产VLBWI住院期间诊治情况比较（中位数±标准差、中位数[M(P25,P75)]）。Table 4 Comparison of diagnosis and treatment of preterm VLBWI during hospitalization between the two groups (median ± standard deviation, median [M(P25, P75)]).
注：☆为Mann Whitney U检验，†为 t 检验，#为卡方检验，※为连续性校正卡方检验。Note: ☆ is the Mann Whitney U test, † is the t test, # is the chi-square test, and ※ is the continuity-corrected chi-square test.
早产VLBWI生后发生BIPI的Logistic多因素回归分析。Logistic multivariate regression analysis of BIPI after birth in preterm VLBWI.
将是否BIPI作为因变量，上述临床资料中行单因素比较后差异有统计学意义的因素作为自变量，进行Logistic多因素回归分析，其结果为：有创通气时间（OR=1.273, 95% CI: 1.056~1.534）、频发呼吸暂停（OR=4.031, 95% CI: 1.355~11.995）、肠外营养时间（OR=1.035, 95% CI: 1.002~1.068）、EUGR（OR=5.883, 95%
CI: 1.984~17.443）为VLBWI生后发生BIPI的独立危险因素，详见表5。Taking BIPI as the dependent variable and the factors with statistically significant differences after univariate comparison in the above clinical data as independent variables, logistic multivariate regression analysis was performed, and the results were: invasive ventilation time (OR=1.273, 95% CI: 1.056-1.534), frequent apnea (OR=4.031, 95% CI: 1.355-11.995), duration of parenteral nutrition (OR=1.035, 95% CI: 1.002-1.068), EUGR (OR=5.883, 95%)
CI: 1.984~17.443) was an independent risk factor for BIPI after VLBWI was born, see Table 5 for details.
表5 早产VLBWI生后发生BIPI的Logistic多因素回归分析结果。Table 5 Logistic multivariate regression analysis results of BIPI after preterm VLBWI.
标本检测：粪便DNA提取（根据试剂盒附带说明书进行）：（1）在室温下，将750μL的裂解液添加到裂解管中，用无菌小勺挖取150mg粪便样本加入裂解管中，使用高频振荡器震荡3min。（2）将裂解管放到离心机里，离心1分钟，将所得上清400μL加到3号柱F中，3号柱F套在一个收集管中，离心1分钟后，将过滤柱丢弃。（3）往上一步得到的收集管中添加1200μL的基因组DNA裂解液并用移液枪吹打以充分混匀。（4）将2号柱套在新的收集管中备用。（5）从步骤（3）中吸取800μL混合液加到2号柱中，离心1分钟，倒掉废液。重复此步骤一次。（6）把2号柱套在一个新的收集管里，添加200μL基因组DNA洗涤液1到2号柱中，在11,000xg下离心1分钟。（此步骤后无需倒掉收集管中的废液，可直接进行下一步。）（7）添加500μL基因组DNA洗涤液2到2号柱中，离心1分钟后，将收集管中的废液倒掉，并将2号柱套回收集管中，再次离心2分钟，尽量除净洗涤液，以免洗涤液中残留乙醇影响下游反应。（8）将2号柱移至干净的收集管中，并打开盖子放置片刻，后添加100μL的基因组DNA洗脱液（事先在65～70℃水浴锅中预热）到柱基质上，室温下放置2~5分钟，离心1分钟洗脱基因组DNA。（9）将抑制物去除柱套在一个收集管中，添加600μL抑制物去除液，离心3分钟。（10）吸取洗脱的基因组DNA100μL放入制备好的抑制物去除柱内，抑制物去除柱套在一个干净的1.5mL离心管中，并离心3分钟，得到的DNA需进行DNA浓度及纯度检测。Specimen detection: Fecal DNA extraction (according to the instructions attached to the kit): (1) At room temperature, add 750 μL of lysis solution to the lysis tube, and use a sterile spoon to pick up 150 mg of the fecal sample and add it to the lysis tube. The frequency oscillator oscillated for 3 min. (2) Put the lysis tube into the centrifuge, centrifuge for 1 minute, add 400 μL of the obtained supernatant to No. 3 column F, cover No. 3 column F in a collection tube, and after centrifugation for 1 minute, discard the filter column. (3) Add 1200 μL of genomic DNA lysis solution to the collection tube obtained in the previous step and mix well by pipetting with a pipette. (4) Put the No. 2 column in a new collection tube for use. (5) Aspirate 800 μL of the mixture from step (3) and add it to the No. 2 column, centrifuge for 1 minute, and discard the waste liquid. Repeat this step once. (6) Put the No. 2 column in a new collection tube, add 200 μL of Genomic DNA Wash 1 to No. 2 column, and centrifuge at 11,000×g for 1 minute. (After this step, there is no need to pour out the waste liquid in the collection tube, you can directly proceed to the next step.) (7) Add 500 μL of Genomic DNA Wash Solution 2 to the No. 2 column, centrifuge for 1 minute, and pour out the waste liquid in the collection tube Then, put the No. 2 column back into the collection tube, centrifuge again for 2 minutes, and try to remove the washing liquid as much as possible, so as to avoid residual ethanol in the washing liquid from affecting the downstream reaction. (8) Move the No. 2 column to a clean collection tube, open the lid and leave it for a while, then add 100 μL of genomic DNA eluate (preheated in a 65-70 °C water bath) to the column matrix, at room temperature. Set aside for 2-5 minutes and centrifuge for 1 minute to elute genomic DNA. (9) Put the inhibitor removal column in a collection tube, add 600 μL of inhibitor removal solution, and centrifuge for 3 minutes. (10) Pipette 100 μL of the eluted genomic DNA into the prepared inhibitor removal column. The inhibitor removal column is sleeved in a clean 1.5 mL centrifuge tube and centrifuged for 3 minutes. The obtained DNA needs to be tested for DNA concentration and purity. .
DNA浓度检测：（1）准备0.5mLEP管。（2）把管子盖子贴上标签。（3）将工作液199μL添加至各个EP管，从提取后的每一个DNA液样品取1μl添加到包含工作液的试管中，然后以2至3秒的涡流混合。（4）所有配置好的样本在室温下孵育2分钟。（5）将孵育好的样本置于QubitTM3.0
Fluorometer上测定DNA浓度，并记录数值。（6）测定完成后将提取成功的DNA液（浓度≥5ng/ul）暂时保存在-20℃的冰箱中备用。DNA concentration detection: (1) Prepare a 0.5mL EP tube. (2) Label the tube cap. (3) Add 199 μL of the working solution to each EP tube, add 1 μL from each DNA liquid sample after extraction to the test tube containing the working solution, and mix by vortexing for 2 to 3 seconds. (4) Incubate all prepared samples for 2 minutes at room temperature. (5) Place the incubated samples in QubitTM3.0
DNA concentration was determined on a Fluorometer and the value recorded. (6) After the measurement is completed, the successfully extracted DNA solution (concentration ≥ 5ng/ul) is temporarily stored in a refrigerator at -20°C for future use.
MetaVx文库构建：文库构建使用金唯智设计的PCR引物扩增原核生物16Sr
RNA基因上V3~V4的2个高度可变区，具体步骤如下：（1）1st PCR 以样品中提取的 DNA稀释至5ng/μL为模板，使用现有试剂盒中的Primer和PCR酶对V3～V4区域进行扩增，使用H2O作为阴性对照同时进行实验。在冰上配制以下试剂。除DNA提取液和H2O以外，依照如下组份先按反应数＋α的量配制扩增预混Mix，取配制好的预混Mix分装到PCR反应管中。反应体系的配置（总体积25μl）: 2×PCR Master Mix (Mg2+, dNTPs plus) 12.5μL、16S V3-V4
Forward Primer（10μM）1μL、16S V3-V4 Reverse Primer（10μM）1μL、Microbial DNA
5ng/μL2.5μL、ddH2O8μL。注：此处不添加DNA提取液。取上面反应管中的一个作为阴性对照，在反应管中加入H2O后将管盖压紧。 添加DNA提取液。 按以下条件进行PCR反应：＜初期变性＞95℃，3分钟；＜PCR：27cycles＞95℃，30秒；60℃，30秒；72℃，20秒；＜总延伸＞72℃，5分钟；＜Hold＞4℃，Hold。1st PCR产物的纯化：使用Agencourt AMPure XP（磁珠）对1st
PCR扩增产物进行纯化。4℃冰箱取出，室温放置30
min，将磁珠充分打散，混匀。扩增产物与磁珠的结合。按以下比例在PCR产物中加入磁珠：PCR产物25μL、Agencourt AMPure
XP45μL。充分混匀，室温孵育5 min，瞬时离心，磁力架上静置3min，去除上清液。 第一次清洗 反应管放置在磁力架上不动，加入200μl新配的70%的乙醇，室温静置1min，去除80%的乙醇。第二次清洗 重复一次上述4）第一次清洗操作，轻轻离心，去除残留70%的乙醇。磁珠的干燥 室温干燥10min。 洗脱 加入40μL的洗脱液，轻轻地上下吸打，充分混匀，室温孵育5min，瞬时离心，磁力架上静置3min，回收上清液。2nd PCR：以操作纯化得到的1st PCR产物为模板，进行2nd PCR导入测序接头，可对检测样品添加不同组合的Index
primer。 在冰上配制以下反应液（在区域1操作）。除1st PCR纯化产物以外，依照如下组份先按反应数+α的量配制扩增预混Mix，取配制好的预混Mix分装到PCR反应管中。 反应体系的配置（总体积50μL）：2×PCR Master Mix
(Mg2+, dNTPs plus) 25μL、Index 1 Primer（10μM）2μL、Index 2
Primer（10μM）2μL、DNA（1st PCR 纯化产物）10μL、ddH2O11μL。注：此处不添加1st PCR纯化产物。添加DNA提取液（在区域 3 操作）。 按以下条件进行PCR反应：＜初期变性＞95℃，3分钟；＜PCR：9cycles＞95℃，30秒；60℃，30秒；72℃，30秒 ；＜总延伸＞72℃，5分钟；＜Hold＞4℃，Hold。2nd PCR产物的纯化：与【1st PCR】产物的纯化进行相同的操作，对2nd PCR产物进行纯化：PCR产物50μL、Agencourt AMPure
XP90μL。文库质检 使用Agilent2100生物分析仪检测文库质量，并且通过QubitTM3.0
Fluorometer检测文库浓度。DNA测序：（1）文库预处理：1）准备变性试剂：取200μL1N文库变性液浓储，加800μL无酶水配置成0.2N文库变性液，涡旋混匀，短暂离心，室温放置备用。2）文库变性：取5μL文库（4nM）至低吸附管管底，再加入5μL0.2N文库变性液，吹打混匀。关闭管盖，室温孵育5min，之后立即置于冰上。3）文库稀释：变性后的10μL文库中加入990μL冰上预冷的稀释缓冲液，混匀后快速离心，置于冰上备用。（2）上样：1）使用洁净的1mL枪头刺穿Load Samples（装入样品）孔的封箔，将600μL已制备文库注入Load Samples孔中。避免接触封箔。2）加载样品后检查孔中是否有气泡，如果有气泡在工作台轻轻敲打试剂盒以释放气泡。 3）将加载好样品的测序试剂盒使用MiSeq测序仪进行2×250bp双端测序（PE），使用MiSeq自带的MiSeq Control
Software读取序列信息。对测序得到的原始数据（Raw Data）进行过滤，去除不合格序列，再进行拼接、去除嵌合体等质检工作，得到有效数据(Clean Data)。MetaVx Library Construction: Library Construction Amplifies Prokaryotic 16Sr Using PCR Primers Designed by Goldwisdom
The two hypervariable regions of V3~V4 on the RNA gene, the specific steps are as follows: (1) 1st PCR Dilute the DNA extracted from the sample to 5ng/μL as a template, and use the Primer and PCR enzymes in the existing kit to perform V3 The ~V4 region was amplified and experiments were performed concurrently using H2O as a negative control. Prepare the following reagents on ice. In addition to DNA extraction solution and H2O, prepare amplification premix Mix according to the following components according to the number of reactions + α, and dispense the prepared premix Mix into PCR reaction tubes. Configuration of the reaction system (total volume 25μl): 2×PCR Master Mix (Mg2+, dNTPs plus) 12.5μL, 16S V3-V4
Forward Primer (10 μM) 1 μL, 16S V3-V4 Reverse Primer (10 μM) 1 μL, Microbial DNA
5ng/μL2.5μL, ddH2O8μL. Note: DNA extraction solution is not added here. Take one of the above reaction tubes as a negative control, add H2O to the reaction tube and press the cap tightly. Add DNA extraction solution. Carry out the PCR reaction under the following conditions: <initial denaturation> 95°C, 3 minutes; <PCR: 27 cycles> 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 20 seconds; <total extension> 72°C, 5 minutes; <Hold>4°C, Hold. Purification of 1st PCR product: Use Agencourt AMPure XP (magnetic beads) for 1st
PCR amplification products were purified. Take it out of the refrigerator at 4°C and leave it at room temperature for 30
min, fully disperse the magnetic beads and mix well. Binding of amplified products to magnetic beads. Add magnetic beads to the PCR product in the following proportions: PCR product 25 μL, Agencourt AMPure
XP 45 μL. Mix well, incubate for 5 min at room temperature, centrifuge briefly, stand on a magnetic stand for 3 min, and remove the supernatant. The first washing The reaction tube was placed on the magnetic stand still, 200 μl of freshly prepared 70% ethanol was added, and the reaction tube was allowed to stand at room temperature for 1 min to remove the 80% ethanol. The second washing Repeat the above 4) first washing operation, and centrifuge gently to remove the residual 70% ethanol. Drying of magnetic beads Dry at room temperature for 10 min. Elution Add 40 μL of eluate, gently pipette up and down, mix well, incubate at room temperature for 5 min, centrifuge briefly, stand on a magnetic stand for 3 min, and recover the supernatant. 2nd PCR: Using the 1st PCR product obtained by the operation and purification as the template, the 2nd PCR is carried out and introduced into the sequencing adapter, and different combinations of Index can be added to the test sample
primer. Prepare the following reactions (operated in Zone 1) on ice. In addition to the 1st PCR purified product, prepare the amplification premix Mix according to the following components according to the number of reactions + α, and dispense the prepared premix Mix into PCR reaction tubes. Configuration of the reaction system (total volume 50 μL): 2×PCR Master Mix
(Mg2+, dNTPs plus) 25μL, Index 1 Primer (10μM) 2μL, Index 2
Primer (10 μM) 2 μL, DNA (1st PCR purified product) 10 μL, ddH2O 11 μL. NOTE: The 1st PCR purified product is not added here. Add DNA extraction solution (operate in section 3). Carry out the PCR reaction under the following conditions: <initial denaturation> 95℃, 3 minutes; <PCR: 9 cycles> 95℃, 30 seconds; 60℃, 30 seconds; 72℃, 30 seconds; <total extension> 72℃, 5 minutes; <Hold>4°C, Hold. Purification of the 2nd PCR product: The same operation as the purification of the [1st PCR] product was performed, and the 2nd PCR product was purified: PCR product 50 μL, Agencourt AMPure
XP 90 μL. Library quality inspection Using Agilent2100 bioanalyzer to detect library quality, and pass QubitTM3.0
Fluorometer detects library concentration. DNA sequencing: (1) Library pretreatment: 1) Prepare denaturation reagents: Take 200 μL of 1N library denaturing solution for concentrated storage, add 800 μL of enzyme-free water to prepare 0.2N library denaturing solution, vortex to mix, centrifuge briefly, and set aside at room temperature for later use. 2) Denaturation of library: Take 5μL of library (4nM) to the bottom of the low adsorption tube, add 5μL of 0.2N library denaturation solution, and mix by pipetting. Close the lid and incubate at room temperature for 5 min, then place on ice immediately. 3) Library dilution: Add 990 μL of ice-cold dilution buffer to the denatured 10 μL library, mix well, centrifuge quickly, and place on ice for later use. (2) Loading: 1) Pierce the sealing foil of the Load Samples well with a clean 1mL pipette tip, and inject 600 μL of the prepared library into the Load Samples well. Avoid touching the sealing foil. 2) After loading the sample, check whether there are air bubbles in the wells, if there are air bubbles, tap the kit lightly on the workbench to release the air bubbles. 3) Use the MiSeq sequencer to perform 2×250bp paired-end sequencing (PE) with the loaded sequencing kit, and use the MiSeq Control that comes with MiSeq
Software reads sequence information. The raw data obtained by sequencing (Raw Data) is filtered to remove unqualified sequences, and then quality inspections such as splicing and removal of chimeras are performed to obtain valid data (Clean Data).
粪便16S rDNA高测序结果分析：测序数据初步统计：共检测两组早产VLBWI粪便样本共708份，其中BIPI组276份，非BIPI组432份，生后第1天粪便样本提取率为18.64%，在分析过程中将提取或建库失败的样本、第一天粪便样本剔除，最终保留两组早产儿粪便样本成功检测数量共392份，BIPI组148份，非BIPI组244份。随机在样本中抽取一定数量的个体，并计算其所代表的物种数目，用这些个体数与物种数构建稀释性曲线，以说明样本测序数据的深度及合理性，曲线趋向平坦时，说明测序深度足够，测序数据越合理，详见图1，稀释性曲线可反映物种丰度和物种均匀度，曲线横轴上的长度反映物种丰度，范围越大，说明物种的丰度越高；曲线的形状反映物种均匀度，曲线越平坦，说明物种的均匀度越高。Analysis of fecal 16S rDNA high sequencing results: Preliminary statistics of sequencing data: A total of 708 fecal samples from two groups of preterm VLBWI were detected, including 276 in the BIPI group and 432 in the non-BIPI group. The extraction rate of fecal samples on the first day after birth was 18.64%. During the analysis, the samples that failed to extract or build the library and the fecal samples on the first day were excluded. Finally, 392 fecal samples were successfully detected in the two groups of premature infants, 148 in the BIPI group and 244 in the non-BIPI group. Randomly select a certain number of individuals from the sample, and calculate the number of species they represent. Use these individual numbers and species numbers to construct a dilution curve to illustrate the depth and rationality of the sample sequencing data. When the curve tends to be flat, it indicates the sequencing depth. enough, the more reasonable the sequencing data is, see Figure 1 for details. The dilution curve can reflect species abundance and species uniformity. The length on the horizontal axis of the curve reflects species abundance. The larger the range, the higher the species abundance. The shape reflects the evenness of the species, and the flatter the curve, the higher the evenness of the species.
OTU聚类分析及注释：操作分类单元（Operational taxonomy unit，OTU）是在生物分析时假定的分类单元。将样品序列中相似性高于97%的序列定义为一个OTU，每个OUT对应一个不同的16S rDNA基因序列，即每个OTU对应一个不同的物种。对所有样本的有效序列，以97%的一致性进行OTU聚类，对OTU序列进行物种注释及物种聚类。为了快速直观的展示样本中的物种组成及丰度信息，因此构建了OTU对应的物种注释Heatmap，展现两组早产儿不同日龄时的OTU丰度信息。根据各样本在不同分层级上物种的相对分度，选取丰度排名前50的物种，在物种和样本层面分别进行聚类分析并绘制Heatmap；通过将不同丰度的物种分块聚集，从颜色梯度上来反应多个样品在各分类水平上群落组成的相似性和差异性。在本实验被测标本中，生后第4天，只有少量同组别样本聚类到一起，且两组样本丰度相仿，详见图2。生后第28天，大部分BIPI组样本聚类到左侧，大部分非BIPI组样本聚类到右侧，表明两组样本区分较为明显，且左侧BIPI组样本丰度较右侧非BIPI组偏高，详见图3。图2、图3中，行名是OTU编号，列名是样本组别，图左侧为OTU聚类树，上方为样本聚类树，中间每一个方格的颜色对应一行OTU相对丰度经过归一化处理（log10）后的值，红色到蓝色的渐变色反映物种丰度由高到低的变化，越接近红色，丰度越高，越接近蓝色，丰度越低。OTU cluster analysis and annotation: Operational taxonomy unit (OTU) is a taxa that is assumed in biological analysis. A sequence with a similarity higher than 97% in the sample sequence was defined as an OTU, and each OUT corresponds to a different 16S rDNA gene sequence, that is, each OTU corresponds to a different species. For the valid sequences of all samples, OTU clustering was performed with 97% consistency, and species annotation and species clustering were performed on the OTU sequences. In order to quickly and intuitively display the species composition and abundance information in the samples, the species annotation Heatmap corresponding to the OTU was constructed to display the OTU abundance information of the two groups of premature infants at different ages. According to the relative distribution of species in each sample at different levels, select the top 50 species in abundance, perform cluster analysis at the species and sample levels respectively, and draw Heatmaps; by clustering species with different abundances, from The color gradient reflects the similarity and difference of the community composition of multiple samples at each taxonomic level. Among the samples tested in this experiment, on the 4th day after birth, only a small number of samples from the same group were clustered together, and the abundance of the two groups of samples was similar, as shown in Figure 2. On the 28th day after birth, most of the samples in the BIPI group were clustered to the left, and most of the samples in the non-BIPI group were clustered to the right, indicating that the two groups were clearly distinguished, and the samples in the left BIPI group were more abundant than those in the right non-BIPI group. The group is high, see Figure 3 for details. In Figure 2 and Figure 3, the row name is the OTU number, and the column name is the sample group. The left side of the figure is the OTU clustering tree, and the top is the sample clustering tree. The color of each square in the middle corresponds to a row of OTU relative abundance. The value after normalization (log10), the gradient color from red to blue reflects the change of species abundance from high to low, the closer to red, the higher the abundance, and the closer to blue, the lower the abundance.
两组肠道菌群物种丰度分析。在门水平上，两组均以厚壁菌门（Firmicutes）、变形菌门（Proteobacteria）为主，其次是拟杆菌门（Bacteroidetes）和放线菌门（Actinobacteria）；在纲水平上，两组以γ-变形菌纲（Gammaproteobacteria）所占比例最高，其次是芽孢杆菌纲（Bacilli）、梭菌纲（Clostridia）、放线菌纲（Actinobacteria）、拟杆菌纲（Bacteroidia）；在科水平上，BIPI组以肠杆菌科(Enterobacteriaceae)、肠球菌科(Enterococcaceae)最为丰富，其次是葡萄球菌科(Staphylococcaceae)、拟杆菌科(Bacteroidaceae)、梭菌科（Clostridiaceae），非BIPI组以肠杆菌科(Enterobacteriaceae)最为丰富，其次肠球菌科、葡萄球菌科、链球菌科(Streptococcaceae) ；在属水平上，两组相对丰度排在前五位的为克雷伯氏菌属（Klebsiella）、肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)、拟杆菌属(Bacteroides)，其中肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)
在两组间的相对丰度差异见图4、图5、图6，可看出具有显著差异，符合统计学意义；克雷伯氏菌属(Klebsiella)、拟杆菌属(Bacteroides)、假单胞菌属
(Pseudomonas) 在两组间的相对丰度差异见图7、图8、图9，可看出不具差异或者差异无规律。可以看出，肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)的结合作为早产儿脑损伤微生物标志物的准确性以及特异性良好，可作为非确诊的辅助信息。Species abundance analysis of gut microbiota between two groups. At the phylum level, the two groups are dominated by Firmicutes and Proteobacteria, followed by Bacteroidetes and Actinobacteria; at the class level, the two groups Gammaproteobacteria has the highest proportion, followed by Bacilli, Clostridia, Actinobacteria and Bacterodia; at the family level, Enterobacteriaceae and Enterococcaceae were the most abundant in the BIPI group, followed by Staphylococcaceae, Bacteroidaceae, and Clostridiaceae. The non-BIPI group was dominated by Enterobacteriaceae ( Enterobacteriaceae is the most abundant, followed by Enterococcus, Staphylococcus, and Streptococcus; at the genus level, the top five relative abundances of the two groups are Klebsiella, Enterobacter Genus (Enterobacteriaceae), Enterococcus (Enterococcus), Staphylococcus (Staphylococcus), Bacteroides (Bacteroides), including Enterobacteriaceae (Enterobacteriaceae), Enterococcus (Enterococcus), Staphylococcus (Staphylococcus)
The relative abundance differences between the two groups are shown in Figure 4, Figure 5, and Figure 6. It can be seen that there is a significant difference, which is in line with statistical significance; Klebsiella, Bacteroides, Pseudomonas Bacteria
The relative abundance difference of (Pseudomonas) between the two groups is shown in Figure 7, Figure 8, and Figure 9. It can be seen that there is no difference or the difference is irregular. It can be seen that the combination of Enterobacteriaceae, Enterococcus, and Staphylococcus has good accuracy and specificity as a microbial marker for brain injury in premature infants, and can be used as non-diagnostic auxiliary information.
Alpha多样性分析：Alpha多样性（Alpha diversity），指一个特定环境或生态系统内的多样性，通过用物种累积箱形图、物种多样性曲线和一系列统计学分析指数来评估各样本中微生物群落物种丰富度和多样性的差异。（1）Chao1指数：用于估计样品中所含OTU数目，数值越大代表物种越多，它对稀有的物种很敏感。（2）observed
species指数：生物信息学分析以OTU为单位，observed
species和Chao1指数都是用来表示样品中的OTU数目，说明物种丰富度。（3）Shannon指数:主要是用来描述OTU出现的紊乱和不确定性，越大的Shannon指数值，个体分布越均。（4）Simpson指数：在生态学中用来定量的描述一个区域的生物多样性。越大的Simpson指数值，预示着越高的群落多样性。Alpha diversity analysis: Alpha diversity (Alpha diversity), refers to the diversity within a specific environment or ecosystem, through the use of species cumulative box plots, species diversity curves and a series of statistical analysis indices to evaluate the microorganisms in each sample Differences in community species richness and diversity. (1) Chao1 index: It is used to estimate the number of OTUs contained in the sample. The larger the value, the more species, and it is very sensitive to rare species. (2) observed
species index: bioinformatics analysis in OTU units, observed
Both species and Chao1 indices are used to represent the number of OTUs in the sample, indicating species richness. (3) Shannon index: It is mainly used to describe the disorder and uncertainty of OTU. The larger the Shannon index value, the more uniform the individual distribution. (4) Simpson index: used in ecology to quantitatively describe the biodiversity of a region. The larger the Simpson index value, the higher the community diversity.
BIPI组chao1指数、observed species指数在生后第4~28天均高于非BIPI组，且在生后21、28天时显著高于非BIPI组（P<0.05），提示BIPI组肠道菌群丰富度在21、28天时较非BIPI组显著增高，详见图10、11；BIPI组shannon指数、simpson指数在生后第4~28天均高于非BIPI，且在生后21天、28天显著高于非BIPI组（P<0.05），提示与非BIPI组相比，BIPI组在第21、28天时肠道菌群多样性显著增多，详见图12、13。横坐标为样本采集时间，纵坐标为各指数，箱子上下两端分别为第75分位数、第25百分位数，箱子中间的横线为中位数，上下触须的末端分别为第95百分位数和第5百分位数，圆点为第95百分位数和第5百分位数以外的数值。*表示P值<0.05。The chao1 index and observed species index of the BIPI group were higher than those of the non-BIPI group on the 4th to 28th days after birth, and were significantly higher than those of the non-BIPI group on the 21st and 28th days after birth (P<0.05), suggesting that the intestinal flora of the BIPI group The richness was significantly higher than that of the non-BIPI group on the 21st and 28th days, as shown in Figures 10 and 11; the BIPI group's shannon index and simpson index were higher than those of the non-BIPI group on the 4th to 28th days after birth, and the 21st and 28th days after birth in the BIPI group days were significantly higher than those in the non-BIPI group (P<0.05), suggesting that the BIPI group had a significantly increased diversity of intestinal flora on days 21 and 28 compared with the non-BIPI group, as shown in Figures 12 and 13. The abscissa is the sample collection time, and the ordinate is each index. The upper and lower ends of the box are the 75th percentile and the 25th percentile, respectively. The horizontal line in the middle of the box is the median, and the ends of the upper and lower tentacles are the 95th percentile. Percentile and 5th percentile, dots are values outside the 95th and 5th percentile. * indicates P value < 0.05.
Beta多样性分析：Beta多样性（Beta diversity）是生物多样性的重要组成部分，目的用来研究不同样品或处理组之间的物种群落结构差异。它与Alpha多样性不同，Beta多样性则是比较不同样品（生态系统）之间的多样性差异，而Alpha多样性主要用于描述单个样品的物种多样性。主成分分析（PCA，Principal
Component Analysis），是应用方差分解，对多维数据进行降维，提取出数据中最主要的元素和结构的方法[51]。主坐标分析（PCoA, Principal Co-ordinates Analysis），是通过一系列的特征值和特征向量排序从多维数据中提取出最主要的元素和结构的方法。PCoA基于距离矩阵，选取贡献率最大的主坐标组合进行作图分析。两组间PCA和PCoA显示，两组之间Beta多样性第21天及第28天差异有统计学意义（P<0.05），同组内部聚集在一起，生后28天BIPI组与非BIPI组之间内部聚集更加明显，见图14，A、C、E为两组4、21、28天PCA情况，B、D、F为两组4、21、28天PCoA情况，不同形状和不同颜色对应图底部相应的分组名称。Beta diversity analysis: Beta diversity is an important part of biodiversity, and it is used to study the differences in species community structure between different samples or treatment groups. It is different from Alpha diversity, which compares the diversity differences between different samples (ecosystems), while Alpha diversity is mainly used to describe the species diversity of a single sample. Principal Component Analysis (PCA, Principal
Component Analysis) is a method of applying variance decomposition to reduce the dimension of multidimensional data and extract the most important elements and structures in the data [51]. Principal Coordinates Analysis (PCoA, Principal Co-ordinates Analysis) is a method of extracting the most important elements and structures from multidimensional data through a series of eigenvalues and eigenvector sorting. Based on the distance matrix, PCoA selects the principal coordinate combination with the largest contribution rate for mapping analysis. PCA and PCoA between the two groups showed that there was a statistically significant difference in the Beta diversity between the two groups on the 21st and 28th days (P<0.05), which were clustered together within the same group, and the BIPI group and the non-BIPI group at 28 days after birth The internal aggregation between the two groups is more obvious, as shown in Figure 14, A, C, E are the PCA conditions of the two groups at 4, 21, and 28 days, and B, D, and F are the PCoA conditions of the two groups at 4, 21, and 28 days, with different shapes and colors. Corresponds to the corresponding group name at the bottom of the graph.
组间菌群结构差异显著性分析：根据样品丰度表，进行样本或样本组间的物种差异分析。在不同时间的点（生后第4、7、14、21、28天）的不同分类水平下（门、纲、目、科、属、种）分别进行差异分析，筛选差异显著物种。本研究利用Qiime分析软件中MetagenomeSeq方法分析筛选两组样本之间具有显著差异的微生物物种，P 值<0.05作为差异显著性筛选阈值。两组间总体，在门水平上，BIPI组变形菌门（Proteobacteria）（P=0.001）显著减少，厚壁菌门（Firmicutes）（P=0.029）、放线菌门（Actinobacteria）（P=0.021）、拟杆菌门（Bacteroidetes）（P=0.016）显著增多；在纲的分类水平上，BIPI组γ-变形菌纲（Gammaproteobacteria）（P=0.001）、梭菌纲（Clostridia）（P=0.018）显著减少，拟杆菌纲（Bacteroidia）（P=0.016）显著增多；在科水平上，BIPI组肠杆菌科（Enterobacteriaceae）（P<0.001）、梭菌科（Clostridiaceae）（P=0.005）显著减少，而肠球菌科（Enterococcaceae）（P=0.001）、拟杆菌科（P=0.001）显著增多。生后第4天，在门水平上，BIPI组变形菌门（P＝0.048）显著减少，而厚壁菌门（P＝0.025）显著增多；在纲水平，BIPI组γ-变形菌纲（P=0.035）显著减少，芽孢杆菌纲明显增加，有统计学意义（P=0.046）；在科水平上，BIPI组肠杆菌科（P＝0.040）、梭菌科（P＝0.025）显著减少；在属的水平上，BIPI组中梭菌属（P＝0.025）显著减少。生后第7天，在门水平，BIPI组变形菌门（P＝0.003）显著减少，厚壁菌门（P＝0.026）、放线菌门（P=0.046）显著增多；在纲水平上，BIPI组γ-变形菌纲（P=0.002）显著减少，芽孢杆菌纲（P=0.012）、放线菌纲（P=0.046）显著增多；在科水平，BIPI肠杆菌科（P=0.001）显著减少；在属的水平上，BIPI组肠杆菌属（P=0.001）、克雷伯杆菌属（P=0.004）显著减少，芽孢菌属显著增多（P=0.012）。生后第14天，在科水平，BIPI组肠杆菌科（P=0.002）显著减少，拟杆菌科（P=0.037）显著增多；在属水平上，肠杆菌属（P=0.002）显著减少、拟杆菌属（P=0.036）显著增多。生后第21天，BIPI组：变形菌门（P=0.001）、放线菌门（P=0.018）显著减少，而拟杆菌门（P<0.001）显著增多；在纲水平上，BIPI组γ-变形菌纲（P=0.001）、梭菌纲（P=0.021）显著减少，而拟杆菌纲（P<0.001）增多；在科水平，肠杆菌科（P=0.003）、梭菌科（P=0.020）显著减少，拟杆菌科显著增多（P<0.001）。生后第28天，BIPI组：在门水平，变形菌门（P=0.001）、防线菌门（p=0.003）显著减少，而厚壁菌门（P=0.001）、拟杆菌门（P=0.001）显著增多；在纲水平上，γ-变形菌纲（P=0.001）、梭菌纲（P=0.012）显著减少，拟杆菌纲（P=0.001）显著增多；在科水平，肠杆菌科（P=0.003）、梭菌科（P=0.012）、双歧杆菌科（P=0.010）显著减少，而肠球菌科（P＝0.0016）、拟杆菌科（P=0.001）显著增多。Significant difference analysis of bacterial community structure between groups: According to the sample abundance table, the species difference analysis between samples or sample groups is carried out. Differential analysis was carried out at different time points (4th, 7th, 14th, 21st, 28th days after birth) and under different classification levels (phylum, class, order, family, genus, species), and the species with significant differences were screened. In this study, the MetagenomeSeq method in Qiime analysis software was used to analyze and screen microbial species with significant differences between the two groups of samples, with a P value of <0.05 as the screening threshold for significant differences. Overall between the two groups, at the phylum level, Proteobacteria (P=0.001) was significantly reduced in the BIPI group, Firmicutes (P=0.029), Actinobacteria (P=0.021) ), Bacteroidetes (P=0.016) increased significantly; at the class classification level, BIPI group γ-Proteobacteria (P=0.001), Clostridia (P=0.018) Significantly decreased, Bacterodia (P=0.016) increased significantly; at the family level, Enterobacteriaceae (P<0.001) and Clostridiaceae (P=0.005) were significantly decreased in BIPI group, While Enterococcaceae (P=0.001) and Bacteroidetes (P=0.001) increased significantly. On the 4th day after birth, at the phylum level, the phylum Proteobacteria (P=0.048) in the BIPI group decreased significantly, while the phylum Firmicutes (P=0.025) increased significantly; =0.035) was significantly reduced, and Bacillus significantly increased, with statistical significance (P=0.046); at the family level, Enterobacteriaceae (P=0.040) and Clostridium (P=0.025) were significantly reduced in the BIPI group; At the genus level, Clostridium was significantly decreased in the BIPI group (P=0.025). On the 7th day after birth, at the phylum level, Proteobacteria (P=0.003) significantly decreased in BIPI group, while Firmicutes (P=0.026) and Actinobacteria (P=0.046) increased significantly; In BIPI group, γ-Proteobacteria (P=0.002) decreased significantly, while Bacillus (P=0.012) and Actinomycetes (P=0.046) increased significantly; at the family level, BIPI Enterobacteriaceae (P=0.001) significantly increased At the genus level, Enterobacter (P=0.001) and Klebsiella (P=0.004) were significantly reduced, and Bacillus was significantly increased (P=0.012) in the BIPI group. On the 14th day after birth, at the family level, Enterobacteriaceae (P=0.002) significantly decreased in BIPI group, and Bacteroidetes (P=0.037) was significantly increased; at the genus level, Enterobacteriaceae (P=0.002) significantly decreased, Bacteroidetes (P=0.036) increased significantly. On the 21st day after birth, in BIPI group: Proteobacteria (P=0.001) and Actinomycetes (P=0.018) decreased significantly, while Bacteroidetes (P<0.001) increased significantly; at the class level, γ in BIPI group - Proteobacteria (P=0.001) and Clostridium (P=0.021) decreased significantly, while Bacteroidetes (P<0.001) increased; at the family level, Enterobacteriaceae (P=0.003), Clostridium (P=0.003) =0.020) was significantly decreased, and Bacteroidetes was significantly increased (P<0.001). On postnatal day 28, BIPI group: at the phylum level, Proteobacteria (P=0.001), Deficitobacteria (P=0.003) were significantly reduced, while Firmicutes (P=0.001), Bacteroidetes (P=0.001) 0.001) significantly increased; at the class level, γ-Proteobacteria (P=0.001), Clostridium (P=0.012) were significantly reduced, and Bacteroidetes (P=0.001) were significantly increased; at the family level, Enterobacteriaceae (P=0.003), Clostridium (P=0.012) and Bifidobacterium (P=0.010) decreased significantly, while Enterococcus (P=0.0016) and Bacteroidetes (P=0.001) increased significantly.
肠道菌群在人类健康和疾病发展中具有重要作用。新生儿期是肠道菌群定植和发展的重要阶段，该时期肠道菌群紊乱会增加未来罹患某些疾病的风险。在本研究中，在胎龄、分娩方式、喂养方式与抗生素使用情况上两组间比较无明显差异，故具有可比性。在生命的早期阶段，肠道菌群的定植与发展可以促进肠道屏障及免疫功能的成熟，并对未来的健康产生深远影响。因此研究早产VLBWI的肠道菌群的特点，探讨其与BIPI的发生是否相关具有重要的意义。The gut microbiota plays an important role in human health and disease development. The neonatal period is an important period for the colonization and development of the gut microbiota, and disturbances in the gut microbiota during this period increase the risk of developing certain diseases in the future. In this study, there were no significant differences between the two groups in terms of gestational age, mode of delivery, feeding mode and antibiotic use, so they were comparable. In the early stages of life, the colonization and development of intestinal flora can promote the maturation of intestinal barrier and immune function, and have a profound impact on future health. Therefore, it is of great significance to study the characteristics of gut microbiota in preterm VLBWI and to explore whether it is related to the occurrence of BIPI.
考虑普通粪便培养受影响因素多，培养成功率低，故本研究采用基于细菌 16S rDNA的V3-V4区的二代高通量测序技术。但在本研究中，生后第一天标本检出率低，可能原因为早产VLBWI生后第一天肠道菌群定植极少，且本研究持续时间较长，标本虽存放于-80℃冰箱，仍有可能被降解，故将第一天样本剔除。Considering that there are many factors affecting common fecal culture and the success rate of culture is low, this study adopted the second-generation high-throughput sequencing technology based on the V3-V4 region of bacterial 16S rDNA. However, in this study, the detection rate of specimens on the first day after birth was low, which may be because the intestinal flora colonization of preterm VLBWI on the first day after birth was very small, and the duration of this study was long, although the samples were stored at -80 ℃ The refrigerator may still be degraded, so the samples on the first day were rejected.
实施例二
：利用本发明肠道微生物辅助诊断早产儿脑损伤的系统，包括测序模块，对分离的肠道菌群核酸样本（粪便DNA）进行测序，以获得测序结果；丰度计算模块，根据测序结果，对肠道菌群中的所述微生物标志物的相对丰度进行检测，得到相对丰度值；比较模块，将得到的微生物标志物相对丰度值与设定值进行比较。设定值为正常组（非BIPI组）得到的微生物标志物相对丰度值。
Example 2 : The system for assisted diagnosis of brain injury in premature infants by using gut microbes of the present invention includes a sequencing module to sequence the isolated gut flora nucleic acid samples (fecal DNA) to obtain sequencing results; the abundance calculation module, according to According to the sequencing result, the relative abundance of the microbial markers in the intestinal flora is detected to obtain the relative abundance value; the comparison module compares the obtained relative abundance value of the microbial marker with the set value. The set value was the relative abundance of microbial markers obtained from the normal group (non-BIPI group).
本发明应用方法简单，无需复杂的数据计算。采集3例出生7日、3例出生4日临床确诊为BIPI且无其他脑部疾病的早产儿粪便样本（非实施例一），分别采用实施例一一样的DNA提取、扩增、测序、OTU物种注释及聚类方法，得到肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)的相对丰度，与实施例一的非BIPI组相比，两组间差异明显，且所有临床对比的差异与实施例一BIPI组近似，倍数误差在10%以内。采集4例出生7日临床确诊为无BIPI但有其疾病（早产儿视网膜病、早产儿呼吸暂停各2例）的早产儿粪便样本，分别采用实施例一一样的DNA提取、扩增、测序、OTU物种注释及聚类方法，得到肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)的相对丰度，与实施例一的非BIPI组相比，所有样本结果差异小，无显著性。The application method of the present invention is simple and does not require complicated data calculation. Stool samples were collected from 3 cases of premature infants who were clinically diagnosed with BIPI and had no other brain diseases on the 7th day and 3 cases were born on the 4th day (not Example 1), and the same DNA extraction, amplification, sequencing, OTU species annotation and clustering methods were used to obtain the relative abundances of Enterobacteriaceae, Enterococcus, and Staphylococcus. Compared with the non-BIPI group in Example 1, there were significant differences between the two groups. , and the differences of all clinical comparisons were similar to those of the BIPI group in Example 1, and the multiple error was within 10%. Stool samples were collected from 4 premature infants who were clinically diagnosed without BIPI on the 7th day of life but had other diseases (2 cases of retinopathy of prematurity, 2 cases of premature infant apnea), and the same DNA extraction, amplification and sequencing as in Example 1 were used respectively. , OTU species annotation and clustering methods to obtain the relative abundance of Enterobacteriaceae, Enterococcus, and Staphylococcus. Compared with the non-BIPI group in Example 1, the results of all samples are different. small, insignificant.
从而，根据肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)与早产儿BIPI的相关性，可以通过检测样本中肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)的丰度来辅助诊断早产儿BIPI，据此本发明公开了一种基于检测肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)丰度的辅助诊断早产儿BIPI的试剂盒。试剂盒组分如下:检测肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)16SrRNA的引物对以及其他常规试剂，比如DNA提取试剂、反应缓冲液、三磷酸碱基脱氧核苷酸、Taq-聚合酶等。Therefore, according to the correlation between Enterobacteriaceae, Enterococcus, Staphylococcus and BIPI in premature infants, it is possible to detect Enterobacteriaceae, Enterococcus, The abundance of Staphylococcus is used to assist in diagnosing BIPI in premature infants. According to this, the present invention discloses an assistant based on detecting the abundance of Enterobacteriaceae, Enterococcus and Staphylococcus. A kit for the diagnosis of BIPI in preterm infants. The kit components are as follows: primer pairs for the detection of Enterobacteriaceae, Enterococcus, Staphylococcus 16SrRNA and other conventional reagents, such as DNA extraction reagents, reaction buffer, triphosphate base deoxygenation Nucleotides, Taq-polymerase, etc.
综上所述，目前对BIPI的发病机制尚未有统一定论，其高危因素包括产前、产时和出生后的各种病理因素。目前研究认为胎龄、出生体重、胎膜早破、绒毛膜炎、窒息是早产儿发生BIPI的产前、产时主要危险因素。本发明公开了肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)在制备早产儿脑损伤微生物标志物中的应用，并根据临床信息，围绕早产儿肠道菌群，探讨了早产VLBWI生命早期肠道菌群定植和BIPI发生、发展过程中肠道菌群的变化，其意义在于公开了新的生物标志物和在早期发育阶段利用微生物群的潜力为BIPI的早期干预提供依据。In summary, there is no unified conclusion on the pathogenesis of BIPI, and its high-risk factors include prenatal, intrapartum and postnatal pathological factors. Current studies suggest that gestational age, birth weight, premature rupture of membranes, chorionitis, and asphyxia are the main prenatal and intrapartum risk factors for BIPI in preterm infants. The invention discloses the application of Enterobacteriaceae, Enterococcus and Staphylococcus in preparing microbial markers of brain injury in premature infants. The colonization of the gut microbiota in the early life of preterm VLBWI and the changes in the gut microbiota during the occurrence and development of BIPI were explored, and the significance of this is to disclose new biomarkers and the potential of using the microbiota at early developmental stages for early intervention of BIPI. Provide evidence.
Claims (10)
- 肠道微生物作为早产儿脑损伤标志物的应用，其特征在于，所述肠道微生物为肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)中的一种或几种。The application of gut microbes as markers of brain injury in premature infants, characterized in that the gut microbes are one or more of Enterobacteriaceae, Enterococcus, and Staphylococcus .
- 肠道微生物在制备早产儿脑损伤标志物中的应用，其特征在于，所述肠道微生物为肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)中的一种或几种。The application of gut microbes in the preparation of premature infant brain injury markers, characterized in that the gut microbes are one of Enterobacteriaceae, Enterococcus, Staphylococcus or several.
- 根据权利要求1或者2所述的应用，其特征在于，早产儿为早产极低出生体重儿。The application according to claim 1 or 2, wherein the premature infant is a premature very low birth weight infant.
- 根据权利要求1或者2所述的应用，其特征在于，所述肠道微生物由肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)组成。The application according to claim 1 or 2, wherein the intestinal microorganisms are composed of Enterobacteriaceae, Enterococcus and Staphylococcus.
- 检测肠道微生物丰度的试剂在制备早产儿脑损伤标志物中的应用；所述肠道微生物为肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)中的一种或几种。Application of a reagent for detecting the abundance of gut microbes in the preparation of markers of brain injury in premature infants; the gut microbes are one of Enterobacteriaceae, Enterococcus, and Staphylococcus or several.
- 根据权利要求5所述的应用，其特征在于，检测肠道微生物丰度的试剂包括检测肠道微生物的引物或者探针。The application according to claim 5, wherein the reagent for detecting the abundance of gut microbes comprises primers or probes for detecting gut microbes.
- 根据权利要求6所述的应用，其特征在于，检测肠道微生物的引物为检测肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)16SrRNA的引物。The application according to claim 6, wherein the primer for detecting intestinal microorganisms is a primer for detecting Enterobacteriaceae (Enterobacteriaceae), Enterococcus (Enterococcus), and Staphylococcus (Staphylococcus) 16SrRNA.
- 利用权利要求1所述肠道微生物辅助诊断早产儿脑损伤的系统，其特征在于，包括测序模块，对分离的肠道菌群核酸样本进行测序，以获得测序结果；丰度计算模块，根据测序结果，对肠道菌群中的所述肠道微生物的相对丰度进行检测，得到相对丰度值；比较模块，将得到的肠道微生物相对丰度值与设定值进行比较。The system for assisting diagnosis of brain injury in premature infants by using the gut microbes according to claim 1, is characterized in that, it includes a sequencing module for sequencing the isolated gut flora nucleic acid samples to obtain sequencing results; an abundance calculation module, according to the sequencing As a result, the relative abundance of the intestinal microorganisms in the intestinal flora is detected to obtain a relative abundance value; the comparison module compares the obtained relative abundance of the intestinal microorganisms with a set value.
- 根据权利要求8所述的系统，其特征在于，设定值为正常组得到的肠道微生物相对丰度值。The system according to claim 8, wherein the set value is the relative abundance value of intestinal microorganisms obtained from the normal group.
- 早产儿脑损伤辅助诊断试剂盒，其特征在于，包括检测肠道微生物的引物或者探针；所述肠道微生物为肠杆菌属（Enterobacteriaceae）、肠球菌属(Enterococcus)、葡萄球菌属(Staphylococcus)中的一种或几种。An auxiliary diagnostic kit for brain injury in premature infants, characterized in that it includes primers or probes for detecting intestinal microorganisms; the intestinal microorganisms are Enterobacteriaceae, Enterococcus, and Staphylococcus. one or more of them.
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|---|---|---|---|---|
| CN108474018A (en) * | 2015-04-16 | 2018-08-31 | 国立研究开发法人国立精神·神经医疗研究中心 | Mental disease biomarker |
| US20200157609A1 (en) * | 2018-10-22 | 2020-05-21 | Virginia Commonwealth University | Salivary and gut microbiota to determine cognitive inpairment |
| CN112760368A (en) * | 2021-01-30 | 2021-05-07 | 苏州大学附属儿童医院 | Application of intestinal microorganisms as premature infant brain injury marker |
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2021
- 2021-02-01 WO PCT/CN2021/074738 patent/WO2022160359A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108474018A (en) * | 2015-04-16 | 2018-08-31 | 国立研究开发法人国立精神·神经医疗研究中心 | Mental disease biomarker |
| US20200157609A1 (en) * | 2018-10-22 | 2020-05-21 | Virginia Commonwealth University | Salivary and gut microbiota to determine cognitive inpairment |
| CN112760368A (en) * | 2021-01-30 | 2021-05-07 | 苏州大学附属儿童医院 | Application of intestinal microorganisms as premature infant brain injury marker |
Non-Patent Citations (1)
| Title |
|---|
| SUNDMAN MARK H.; CHEN NAN-KUEI; SUBBIAN VIGNESH; CHOU YING-HUI: "The bidirectional gut-brain-microbiota axis as a potential nexus between traumatic brain injury, inflammation, and disease", BRAIN, BEHAVIOR AND IMMUNITY., ACADEMIC PRESS, SAN DIEGO, CA, US, vol. 66, 1 January 1900 (1900-01-01), US , pages 31 - 44, XP085228065, ISSN: 0889-1591, DOI: 10.1016/j.bbi.2017.05.009 * |
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