16S rRNA基因分子技术研究仔猪胃肠道菌群区系变化
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摘要
哺乳动物的胃肠道中栖息着大量的微生物,这些微生物在营养、免疫、抗感染等方面对宿主的健康发挥重要作用.仔猪因为要经历早期断奶这样一个特殊的生理阶段,因此,胃肠道微生物对其肠道的健康更显重要.本论文利用基于16S rRNA基因的分子技术研究了仔猪哺乳阶段至断奶后两周(35日龄)的胃肠道菌群区系变化.利用变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)指纹图谱及序列分析技术研究了断奶前、后仔猪胃、空肠和回肠的菌群变化,real-time PCR技术分析了其中一些优势菌群的数量变化;同时,对仔猪小肠食糜与肠壁中的细菌及乳酸杆菌菌群也进行了比较;采用代表性差异分析(Representational difference analysis,RDA)技术还比较了两株来自不同地区仔猪肠道的优势乳酸杆菌——Lactobacillus sobrius菌株;此外,还利用分子技术研究了口服L.sobrius S1后仔猪后肠菌群的变化;最后,基于不同可变区片段对不同乳酸杆菌的16S rRNA基因进行了序列比对分析,以解释一些分子技术的缺陷.
1仔猪哺乳期和断奶后胃中总细菌和乳酸杆菌变化
利用基于16S rRNA基因的PCR/DGGE技术研究了新生仔猪从7日龄至35日龄(断奶后两周)胃中细菌及乳酸杆菌的菌群变化.结果表明,哺乳阶段,仔猪胃食糜维持较低的pH值,断奶后,pH值显著上升;Dilution-PCR半定量分析发现,断奶前,仔猪胃中细菌数量很低,而断奶后,总细菌数量显著上升,乳酸杆菌数量则略有上升;DGGE图谱显示,断奶前胃中细菌条带较单一,优势菌为高迁移条带相关细菌,断奶后图谱中出现一些新的优势条带,而断奶前的优势条带消失;乳酸杆菌DGGE图谱相似性分析显示,断奶前、后仔猪胃中乳酸杆菌菌群变化较小.
2断奶前、后仔猪胃和前肠菌群的变化
本章研究了仔猪从哺乳至断奶后胃、空肠和回肠中菌群的变化.DGGE图谱分析显示,断奶前仔猪前肠图谱中的一些优势条带在断奶后消失,16S rRNA基因序列分析表明与这些条带相对应的细菌分别为L.reuteri,L.johnsonii和L.sobrius;此时,一些新的优势条带出现,序列分析对其鉴定表明,它们主要为Peptostreptococcusanaerobius,Moraxella cuniculi,Streptococcus suis和Porphyromonas catoniae.Real-timePCR定量分析表明,仔猪断奶后,前肠中乳酸杆菌和L.sobrius(断奶前的优势菌)在总细菌中的比例显著下降.同时,建立了S.suis特异性real-time PCR,它能够对猪肠道复杂菌群中的S.suis进行精确定量,最低检测浓度为每微升核酸样品10个16SrRNA基因拷贝数.该分析结果显示,断奶后3天仔猪胃中的S.suis数量达10~7数量级,而在哺乳阶段则未能检测到.
3仔猪空、回肠食糜与肠壁中细菌及乳酸杆菌菌群比较
利用16S rRNA基因分子方法比较了7日龄至35日龄仔猪空、回肠食糜与肠壁中细菌及乳酸杆菌菌群差异.DGGE分析显示,哺乳阶段仔猪空肠食糜的优势菌L.sobrius也在肠壁菌群中出现,而断奶后,空肠食糜中新出现的优势菌S.suis未在肠壁菌群中出现.这一结果在real-timePCR定量分析中也得到证实,所有肠壁样品均为检测到S.suis.比较食糜和肠壁乳酸杆菌菌群DGGE图谱发现,一些特殊条带只在肠壁样品中存在.然而,序列分析显示与这些条带最相近的细菌为alfa-proteobacteria(Brevundimonas vesicularis).对乳酸杆菌特异引物(Lab-0159)与特殊条带相关的细菌的相应序列进行比对发现,它们之间仅有1-2个碱基的错配.
4代表性差异分析比较两株来自不同地区的猪源L.sobrius菌株
代表性差异分析技术近来被引入用于比较高相似性菌株之间的基因组差异,该技术能够在菌株水平对两株细菌基因组进行差异分析.利用DGGE和序列分析技术研究发现来自不同地区的猪源Lactobacillus sp.S1和Lactobacillus sobrius 001~T菌株的16SrRNA基因全序列有很高相似性,大于99%.L.sobrius特异性引物也可以对这两株菌的16S rRNA基因的特定片段进行PCR扩增.代表性差异分析显示,这两株菌的基因组之间未有差异.以上结果说明,Lactobacillus sp.S1菌株也属于L.sobrius,并且推测这两株菌为同一菌株.
5益生菌L.sobrius S1对仔猪后肠菌群的影响
结合PCR-DGGE、16S rRNA基因序列分析及定量real-time PCR技术,研究口服益生菌L.sobrius S1菌株后仔猪从7至35日龄(断奶后两周)后肠菌群的变化.六窝新生仔猪被随机分成两组:对照组和处理组,处理组仔猪于7、9和11日龄口服S1菌液(活菌数5×10~9 CFU·ml~(-1)).分别于7、14、21、24和35日龄,每窝随机屠宰一头仔猪,收集肠道样品.比较不同日龄仔猪后肠菌群DGGE图谱表明,断奶后图谱中多数高迁移距离条带消失,至断奶后两周又逐渐出现.序列分析显示,这些高迁移距离条带主要为乳酸杆菌.平板计数以及定量real-time PCR分析也得到一致的结果.乳酸杆菌特异性PCR-DGGE分析显示,仔猪断奶后,L.amylovorus和L.sobrius从优势乳酸杆菌菌群中消失.通过比较处理组和对照组图谱发现,处理组在14日龄出现一特异条带,与其匹配的序列的最相似已知菌为Clostridium disporicum,相似性为95%;而35日龄时对照组有一特异优势条带,该条带被鉴定为猪链球菌,相似性为99%.L.sobrius特异性real-time PCR分析显示,断奶后3天该菌从仔猪结肠中消失,而处理组仔猪有更早出现该菌的趋势.
6基于不同可变区片段比较乳酸杆菌间16S rRNA基因序列差异
基于16S rRNA基因的PCR-DGGE技术已广泛用于各种环境中菌群生态的研究.近来,一些研究者发现该技术存在一些缺陷,如图谱中的单一条带常常被鉴定为几种细菌,而不同条带会被鉴定为同一种细菌.本研究中,以不同乳酸杆菌为例,分别在三个不同可变区片段比较了不同种乳酸杆菌的16S rRNA基因差异.结果表明,属于不同种的乳酸杆菌之间有相同的16S rRNA基因片段序列;不同可变区片段间,乳酸杆菌16S rRNA基因序列的俩俩间的同源性也有很大差异.两个Lactobacillusamylovorus相关克隆虽然有相同的V2-V3可变区,但是它们V6-V8可变区序列的距离为0.061.这些结果说明,DGGE的这一缺陷很大程度上是由细菌16S rRNA基因序列的自身特性所引起的;结果同时也提示,在设计用于DGGE的PCR引物时,除了考虑引物的特异性外,还应该注意所扩增出的不同细菌的产物序列之间的差异程度.
The mammalian gastrointestinal(GI) tract habors a dense and complex microbiota, which plays an important role in nutrition,immunity and physiology of host.More attention should be paid to the study on the GI microbiota of piglet since it will suffer the special physiological phase like early-weanlng.This present study was carried out to investigate the changes of microbioia in GI tract of piglets from 7 to 35(two weeks after weaning) days of age by using 16S ribosomal RNA(rRNA) gene based molecular methods. Changes in bacterial community composition in stomach,jejunum and ileum of piglets after weaning was investigated using denaturing gradient gel electrophoresis(DGGE),and real-time PCR assay was also used to quantitatively analyze changes in predominant bacterial community.Bacterial and Lactobacillus community between digestia and mucosa samples in jejunum and ileum of piglets were also compared in this thesis.Representational difference analysis(RDA) was used to compare two L.sobrius strains isolated from piglets in different area.16S rRNA-based methods were also used to monitor changes in the hindgut bacterial community of piglets after oral administration of L.sobrius S1.Finally,to explain the limitation of 16S rRNA gene-based methods,sequences alignment analysis was performed to compare 16S rRNA genes of different Lactobacillus spp.based on different variable region fragments.
1 PCR-DGGE analysis of stomach total bacterial and lactobacilli community in sucking and weaned piglets
Molecular methods based on the 16S rRNA gene were used to study bacterial and lactobacilli communities in stomach of neonatal piglets from 7 to 35(two weeks after weaning) days of age.The results showed stomach pH value was about 3.0 before weaning, while the pH value increased apparently after weaning.The semi-quantitative dilution-PCR showed that bacterial concentration in stomach of piglet was very low before weaning, while increased significantly after weaning.Compared with total bacteria,lactobacilli concentration increased slightly.The DGGE profiles from total bacteria in stomach showed the bands of samples before weaning were very simple and the predominant band belonged to far-migrating bacterium.After weaning,some new predominant bands appeared and the previous far-migrating bands disappeared simultaneously.The similarity of DGGE profiles from stomach lactobacilli showed weaning stress had no obvious effect on lactobacilli community.
2 Changes of microbiota in stomach,jejunum and ileum of piglets after weaning
Changes of bacterial community composition in stomach,jejunum and ileum of piglets after weaning(21 day) were investigated by 16S ribosomal RNA(rRNA) gene-based molecular methods.Dehaturing gradient gel electrophoresis(DGGE) profiles from bacterial community in foregut of piglets showed that,after weaning,predominant bands related to Lactobacillus spp.disappeared and substituted by some potential pathogenic species such as Peptostreptococcus anaerobius,Moraxella cuniculi,S.suis and Porphyromonas catoniae. Real-time PCR assay revealed that the percentages of lactobacilli and Lactobacillus sobrius relative to total bacteria were significantly lower in foregut samples of weaned piglets compared with the 21-day piglets.Here,we also described S.suis-specific real-time PCR assay,which can quantify S.suis in complex gastrointestinal microbiota with the 16S rRNA gene concentration higher than 10 copies per microliter DNA sample.This assay showed that S.suis predominated in stomach samples of weaned piglets with population levels up to 10~7 cells per gram of digesta,although this species was not detected in stomach of pre-weaning piglets.
3 Comparison of bacterial and Lactobacillus community between digestia and mucosa samples in jejunum and ileum of piglets
To investigate the differences of bacterial and Lactobacillus community between digesta and mucosa samples in jejunum and ileum of piglets from 7 to 35 days of age,using 16S rRNA gene based methods.DGGE profiles from bacterial community showed that the L.sobrius-related band was also observed in jejunal mucosa of piglets,while the predominant band related to S.suis observed only for weaned piglets did not appear in profiles generated from mucosa samples.Primers(Bact-0027/Lab-0677, Lab-0159/Uiv-0515) were used to amplify the Lactobacillus-specific 16S rRNA gene fragments from digesta and mucosa samples,the PCR products were then analyzed by DGGE.Specific bands were observed in DGGE profiles derived from mucosa-associated bacteria.However,16S rRNA gene sequence analysis indicated that the bands were most closely related to alfa-proteobacteria(Brevundimonas vesicularis).Further sequence alignment revealed that there were one or two bases mismatches between Lactobacillus-specific primers(Lab-0159) and target sequences of 16S rRNA gene from the bands related to alfa-proteobacteria.
4 Comparison of two Lactobacillus strains isolated from piglets in different area by using representational difference analysis
Representational difference analysis(RDA) has been adapted recently to study the genomic diversity of bacterial strains.Two Lactobacillus strains(L.sobrius 001~T and Lactobacillus sp.S1) were isolated from the intestine of piglets in difference area.Highly similarity of 16S rRNA gene between the two strains was found by DGGE and sequencing analysis.Both of these two strains could be detected in 1.2%agarose gel by Lactobacillus sobrius-specific PCR assay.RDA showed that genomic DNA of the two strains was digested with CfoⅠ,similar profiles of DNA fragments were observed between the strains. After subtractive hybridization,no RDA amplified products were found,which suggested that there was no genomic difference between these two strains.The results indicated that strain S1 also belonged to L.sobrius,strain 001~T and S1 were the similar strain.
5 16S ribosomal RNA-based methods to monitor changes in the hindgut bacterial community of piglets after oral administration of L. sobrius S1
Changes in the composition of microbiota in the hindgut of piglets were studied after oral administration of L.sobrius S1,using molecular techniques based on 16S rRNA genes. Six litters of neonatal piglets were randomly divided into control and treatment groups.At 7, 9 and 11 days of age,piglets in the treatment group orally received a preparation of strain S1.V6-V8 and V2-V3 variable regions of the total bacterial and Lactobacillus-specific 16S rRNA gene pool,respectively,were amplified by PCR and analyzed by denaturing gradient gel electrophoresis.DGGE analysis showed that several populations present in the hindgut of piglets,represented by far-migrating bands,disappeared after weaning.Sequence analysis indicated that most of these bands corresponded to Lactobacillus spp.This trend could also be confirmed by plate counting and quantitative real-time PCR.Drastic changes of L.amylovorus and L.sobrius in total lactobacilli populations were also observed in the colon of piglets around weaning,as monitored by Lactobacillus-specific PCR-DGGE. Comparison of DGGE profiles between control and treatment groups revealed a specific band related to Clostridium disporicum that was found in treatment group on day 14.On day 35,a specific band appeared only in the control group,representing a population most closely related to S.suis(99%).Species-specific real-time PCR revealed that the population of L.sobrius which has the same position as strain S1 in DGGE profiles declined apparently in the colon of piglets after weaning,while it tended to come back earlier after oral administration of strain S1.These results indicated that specific populations(mainly Lactobacillus spp.) disappeared from the predominant bacterial community in the porcine hindgnt after weaning.Oral administration of strain S1 could mitigate this disruption, thereby contributing to the prevention of pathogen overgrowth during the critical phase of weaning.
6 Sequences alignment of Lactobacillus spp.16S ribosomal RNA genes based on different variable region fragments
PCR- DGGE based on 16S rRNA gene is commonly used to examine bacterial communities in a variety of environments.However,some unexpected results were observed in recent studies.For example,a single band can be identified to several bacterial species,and several different bands identified to the identical species.Here,we aligned the 16S rRNA gene sequences based on different variable region fragments using Lactobacillus spp.as reference strains.The results showed some different Lactobacillus species possessed the identical partial 16S rRNA gene sequences.Based on different variable region fragments,remarkable difference was observed on pairwise distance in the reference 16S rRNA gene sequences.0.061 of divergence value was found in V6-V8 region of 16S rRNA gene sequences between two L.arnylovorus clones,although they had the identical V2-V3 variable regions.These results indicated that the limitation of DGGE was to some extent due to the characteristic of bacterial 16S rRNA gene sequences.The results also indicated that more attention should be paid to the sequence difference of PCR products among different species,as well as to the specificity of primers when designing new primers for DGGE-PCR.
1仔猪哺乳期和断奶后胃中总细菌和乳酸杆菌变化
利用基于16S rRNA基因的PCR/DGGE技术研究了新生仔猪从7日龄至35日龄(断奶后两周)胃中细菌及乳酸杆菌的菌群变化.结果表明,哺乳阶段,仔猪胃食糜维持较低的pH值,断奶后,pH值显著上升;Dilution-PCR半定量分析发现,断奶前,仔猪胃中细菌数量很低,而断奶后,总细菌数量显著上升,乳酸杆菌数量则略有上升;DGGE图谱显示,断奶前胃中细菌条带较单一,优势菌为高迁移条带相关细菌,断奶后图谱中出现一些新的优势条带,而断奶前的优势条带消失;乳酸杆菌DGGE图谱相似性分析显示,断奶前、后仔猪胃中乳酸杆菌菌群变化较小.
2断奶前、后仔猪胃和前肠菌群的变化
本章研究了仔猪从哺乳至断奶后胃、空肠和回肠中菌群的变化.DGGE图谱分析显示,断奶前仔猪前肠图谱中的一些优势条带在断奶后消失,16S rRNA基因序列分析表明与这些条带相对应的细菌分别为L.reuteri,L.johnsonii和L.sobrius;此时,一些新的优势条带出现,序列分析对其鉴定表明,它们主要为Peptostreptococcusanaerobius,Moraxella cuniculi,Streptococcus suis和Porphyromonas catoniae.Real-timePCR定量分析表明,仔猪断奶后,前肠中乳酸杆菌和L.sobrius(断奶前的优势菌)在总细菌中的比例显著下降.同时,建立了S.suis特异性real-time PCR,它能够对猪肠道复杂菌群中的S.suis进行精确定量,最低检测浓度为每微升核酸样品10个16SrRNA基因拷贝数.该分析结果显示,断奶后3天仔猪胃中的S.suis数量达10~7数量级,而在哺乳阶段则未能检测到.
3仔猪空、回肠食糜与肠壁中细菌及乳酸杆菌菌群比较
利用16S rRNA基因分子方法比较了7日龄至35日龄仔猪空、回肠食糜与肠壁中细菌及乳酸杆菌菌群差异.DGGE分析显示,哺乳阶段仔猪空肠食糜的优势菌L.sobrius也在肠壁菌群中出现,而断奶后,空肠食糜中新出现的优势菌S.suis未在肠壁菌群中出现.这一结果在real-timePCR定量分析中也得到证实,所有肠壁样品均为检测到S.suis.比较食糜和肠壁乳酸杆菌菌群DGGE图谱发现,一些特殊条带只在肠壁样品中存在.然而,序列分析显示与这些条带最相近的细菌为alfa-proteobacteria(Brevundimonas vesicularis).对乳酸杆菌特异引物(Lab-0159)与特殊条带相关的细菌的相应序列进行比对发现,它们之间仅有1-2个碱基的错配.
4代表性差异分析比较两株来自不同地区的猪源L.sobrius菌株
代表性差异分析技术近来被引入用于比较高相似性菌株之间的基因组差异,该技术能够在菌株水平对两株细菌基因组进行差异分析.利用DGGE和序列分析技术研究发现来自不同地区的猪源Lactobacillus sp.S1和Lactobacillus sobrius 001~T菌株的16SrRNA基因全序列有很高相似性,大于99%.L.sobrius特异性引物也可以对这两株菌的16S rRNA基因的特定片段进行PCR扩增.代表性差异分析显示,这两株菌的基因组之间未有差异.以上结果说明,Lactobacillus sp.S1菌株也属于L.sobrius,并且推测这两株菌为同一菌株.
5益生菌L.sobrius S1对仔猪后肠菌群的影响
结合PCR-DGGE、16S rRNA基因序列分析及定量real-time PCR技术,研究口服益生菌L.sobrius S1菌株后仔猪从7至35日龄(断奶后两周)后肠菌群的变化.六窝新生仔猪被随机分成两组:对照组和处理组,处理组仔猪于7、9和11日龄口服S1菌液(活菌数5×10~9 CFU·ml~(-1)).分别于7、14、21、24和35日龄,每窝随机屠宰一头仔猪,收集肠道样品.比较不同日龄仔猪后肠菌群DGGE图谱表明,断奶后图谱中多数高迁移距离条带消失,至断奶后两周又逐渐出现.序列分析显示,这些高迁移距离条带主要为乳酸杆菌.平板计数以及定量real-time PCR分析也得到一致的结果.乳酸杆菌特异性PCR-DGGE分析显示,仔猪断奶后,L.amylovorus和L.sobrius从优势乳酸杆菌菌群中消失.通过比较处理组和对照组图谱发现,处理组在14日龄出现一特异条带,与其匹配的序列的最相似已知菌为Clostridium disporicum,相似性为95%;而35日龄时对照组有一特异优势条带,该条带被鉴定为猪链球菌,相似性为99%.L.sobrius特异性real-time PCR分析显示,断奶后3天该菌从仔猪结肠中消失,而处理组仔猪有更早出现该菌的趋势.
6基于不同可变区片段比较乳酸杆菌间16S rRNA基因序列差异
基于16S rRNA基因的PCR-DGGE技术已广泛用于各种环境中菌群生态的研究.近来,一些研究者发现该技术存在一些缺陷,如图谱中的单一条带常常被鉴定为几种细菌,而不同条带会被鉴定为同一种细菌.本研究中,以不同乳酸杆菌为例,分别在三个不同可变区片段比较了不同种乳酸杆菌的16S rRNA基因差异.结果表明,属于不同种的乳酸杆菌之间有相同的16S rRNA基因片段序列;不同可变区片段间,乳酸杆菌16S rRNA基因序列的俩俩间的同源性也有很大差异.两个Lactobacillusamylovorus相关克隆虽然有相同的V2-V3可变区,但是它们V6-V8可变区序列的距离为0.061.这些结果说明,DGGE的这一缺陷很大程度上是由细菌16S rRNA基因序列的自身特性所引起的;结果同时也提示,在设计用于DGGE的PCR引物时,除了考虑引物的特异性外,还应该注意所扩增出的不同细菌的产物序列之间的差异程度.
The mammalian gastrointestinal(GI) tract habors a dense and complex microbiota, which plays an important role in nutrition,immunity and physiology of host.More attention should be paid to the study on the GI microbiota of piglet since it will suffer the special physiological phase like early-weanlng.This present study was carried out to investigate the changes of microbioia in GI tract of piglets from 7 to 35(two weeks after weaning) days of age by using 16S ribosomal RNA(rRNA) gene based molecular methods. Changes in bacterial community composition in stomach,jejunum and ileum of piglets after weaning was investigated using denaturing gradient gel electrophoresis(DGGE),and real-time PCR assay was also used to quantitatively analyze changes in predominant bacterial community.Bacterial and Lactobacillus community between digestia and mucosa samples in jejunum and ileum of piglets were also compared in this thesis.Representational difference analysis(RDA) was used to compare two L.sobrius strains isolated from piglets in different area.16S rRNA-based methods were also used to monitor changes in the hindgut bacterial community of piglets after oral administration of L.sobrius S1.Finally,to explain the limitation of 16S rRNA gene-based methods,sequences alignment analysis was performed to compare 16S rRNA genes of different Lactobacillus spp.based on different variable region fragments.
1 PCR-DGGE analysis of stomach total bacterial and lactobacilli community in sucking and weaned piglets
Molecular methods based on the 16S rRNA gene were used to study bacterial and lactobacilli communities in stomach of neonatal piglets from 7 to 35(two weeks after weaning) days of age.The results showed stomach pH value was about 3.0 before weaning, while the pH value increased apparently after weaning.The semi-quantitative dilution-PCR showed that bacterial concentration in stomach of piglet was very low before weaning, while increased significantly after weaning.Compared with total bacteria,lactobacilli concentration increased slightly.The DGGE profiles from total bacteria in stomach showed the bands of samples before weaning were very simple and the predominant band belonged to far-migrating bacterium.After weaning,some new predominant bands appeared and the previous far-migrating bands disappeared simultaneously.The similarity of DGGE profiles from stomach lactobacilli showed weaning stress had no obvious effect on lactobacilli community.
2 Changes of microbiota in stomach,jejunum and ileum of piglets after weaning
Changes of bacterial community composition in stomach,jejunum and ileum of piglets after weaning(21 day) were investigated by 16S ribosomal RNA(rRNA) gene-based molecular methods.Dehaturing gradient gel electrophoresis(DGGE) profiles from bacterial community in foregut of piglets showed that,after weaning,predominant bands related to Lactobacillus spp.disappeared and substituted by some potential pathogenic species such as Peptostreptococcus anaerobius,Moraxella cuniculi,S.suis and Porphyromonas catoniae. Real-time PCR assay revealed that the percentages of lactobacilli and Lactobacillus sobrius relative to total bacteria were significantly lower in foregut samples of weaned piglets compared with the 21-day piglets.Here,we also described S.suis-specific real-time PCR assay,which can quantify S.suis in complex gastrointestinal microbiota with the 16S rRNA gene concentration higher than 10 copies per microliter DNA sample.This assay showed that S.suis predominated in stomach samples of weaned piglets with population levels up to 10~7 cells per gram of digesta,although this species was not detected in stomach of pre-weaning piglets.
3 Comparison of bacterial and Lactobacillus community between digestia and mucosa samples in jejunum and ileum of piglets
To investigate the differences of bacterial and Lactobacillus community between digesta and mucosa samples in jejunum and ileum of piglets from 7 to 35 days of age,using 16S rRNA gene based methods.DGGE profiles from bacterial community showed that the L.sobrius-related band was also observed in jejunal mucosa of piglets,while the predominant band related to S.suis observed only for weaned piglets did not appear in profiles generated from mucosa samples.Primers(Bact-0027/Lab-0677, Lab-0159/Uiv-0515) were used to amplify the Lactobacillus-specific 16S rRNA gene fragments from digesta and mucosa samples,the PCR products were then analyzed by DGGE.Specific bands were observed in DGGE profiles derived from mucosa-associated bacteria.However,16S rRNA gene sequence analysis indicated that the bands were most closely related to alfa-proteobacteria(Brevundimonas vesicularis).Further sequence alignment revealed that there were one or two bases mismatches between Lactobacillus-specific primers(Lab-0159) and target sequences of 16S rRNA gene from the bands related to alfa-proteobacteria.
4 Comparison of two Lactobacillus strains isolated from piglets in different area by using representational difference analysis
Representational difference analysis(RDA) has been adapted recently to study the genomic diversity of bacterial strains.Two Lactobacillus strains(L.sobrius 001~T and Lactobacillus sp.S1) were isolated from the intestine of piglets in difference area.Highly similarity of 16S rRNA gene between the two strains was found by DGGE and sequencing analysis.Both of these two strains could be detected in 1.2%agarose gel by Lactobacillus sobrius-specific PCR assay.RDA showed that genomic DNA of the two strains was digested with CfoⅠ,similar profiles of DNA fragments were observed between the strains. After subtractive hybridization,no RDA amplified products were found,which suggested that there was no genomic difference between these two strains.The results indicated that strain S1 also belonged to L.sobrius,strain 001~T and S1 were the similar strain.
5 16S ribosomal RNA-based methods to monitor changes in the hindgut bacterial community of piglets after oral administration of L. sobrius S1
Changes in the composition of microbiota in the hindgut of piglets were studied after oral administration of L.sobrius S1,using molecular techniques based on 16S rRNA genes. Six litters of neonatal piglets were randomly divided into control and treatment groups.At 7, 9 and 11 days of age,piglets in the treatment group orally received a preparation of strain S1.V6-V8 and V2-V3 variable regions of the total bacterial and Lactobacillus-specific 16S rRNA gene pool,respectively,were amplified by PCR and analyzed by denaturing gradient gel electrophoresis.DGGE analysis showed that several populations present in the hindgut of piglets,represented by far-migrating bands,disappeared after weaning.Sequence analysis indicated that most of these bands corresponded to Lactobacillus spp.This trend could also be confirmed by plate counting and quantitative real-time PCR.Drastic changes of L.amylovorus and L.sobrius in total lactobacilli populations were also observed in the colon of piglets around weaning,as monitored by Lactobacillus-specific PCR-DGGE. Comparison of DGGE profiles between control and treatment groups revealed a specific band related to Clostridium disporicum that was found in treatment group on day 14.On day 35,a specific band appeared only in the control group,representing a population most closely related to S.suis(99%).Species-specific real-time PCR revealed that the population of L.sobrius which has the same position as strain S1 in DGGE profiles declined apparently in the colon of piglets after weaning,while it tended to come back earlier after oral administration of strain S1.These results indicated that specific populations(mainly Lactobacillus spp.) disappeared from the predominant bacterial community in the porcine hindgnt after weaning.Oral administration of strain S1 could mitigate this disruption, thereby contributing to the prevention of pathogen overgrowth during the critical phase of weaning.
6 Sequences alignment of Lactobacillus spp.16S ribosomal RNA genes based on different variable region fragments
PCR- DGGE based on 16S rRNA gene is commonly used to examine bacterial communities in a variety of environments.However,some unexpected results were observed in recent studies.For example,a single band can be identified to several bacterial species,and several different bands identified to the identical species.Here,we aligned the 16S rRNA gene sequences based on different variable region fragments using Lactobacillus spp.as reference strains.The results showed some different Lactobacillus species possessed the identical partial 16S rRNA gene sequences.Based on different variable region fragments,remarkable difference was observed on pairwise distance in the reference 16S rRNA gene sequences.0.061 of divergence value was found in V6-V8 region of 16S rRNA gene sequences between two L.arnylovorus clones,although they had the identical V2-V3 variable regions.These results indicated that the limitation of DGGE was to some extent due to the characteristic of bacterial 16S rRNA gene sequences.The results also indicated that more attention should be paid to the sequence difference of PCR products among different species,as well as to the specificity of primers when designing new primers for DGGE-PCR.
引文
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