Eukaryotic food sources analysis in situ of tropical common sea cucumber Holothuria leucospilota based on 18S rRNA gene high-throughput sequencing

Sea cucumber Holothuria leucospilota is one of the most widespread tropical holothurian species.In this study,eukaryotic organism composition in foregut and hindgut contents of H.leucospilota and surrounding sediments was assessed by 18S rRNA gene high-throughput sequencing.Eukaryon richness and diversity in the habitat sediments were significantly higher than those in foregut and hindgut contents of the sea cucumbers(P<0.05).The foregut content group,hindgut content group,and marine sediment group sequences were respectively assigned to 18.20±1.32,19.40±1.03,and 21.80±0.37 phyla.In the foregut contents,Nematoda(20.18%±9.59%),Mollusca(16.12%±10.49%),Chlorophyta(10.04%±4.85%),Annelida(8.72%±10.93%),Streptophyta(8.46%±4.65%),and Diatomea(5.99%±2.01%)were the predominant phyla,which showed the eukaryotic food sources of H.leucospilota were primarily belong to the above phyla.The predominant phyla in the hindgut contents were Streptophyta(45.55%±17.32%),Mollusca(4.93%±4.82%),Arthropoda(5.37%±3.08%),Diatomea(3.88%±2.34%),and Chlorophyta(3.79%±1.59%);and Annelida(37.80%±17.00%),Arthropoda(24.49%±12.53%),Platyhelminthes(7.14%±3.02%),Nematoda(4.14%±0.91%),and Diatomea(5.11%±1.35%)had large contents in the sediments.The comparatively high content of Paris genus in phylum Streptophyta in foregut contents indicated that land plants were one of the primary food sources of H.leucospilota,however the significantly higher contents of Streptophyta in hindgut contents than that in foregut contents might suggest a large part of the terrigenous detritus ingested might not be digested by H.leucospilota.UPGMA and PCoA analysis revealed that eukaryotic organism composition differed significantly between foregut contents of H.leucospilota and ambient sediments,indicating selective feeding feature of H.leucospilota.This study provided useful references for artificial feed of tropical sea cucumbers and enhanced understanding of the ecological roles of detritus-feeding macrobenthos.

陕西省榆林地区鸡蛔虫核糖体18S rRNA与ITS-2序列分析及种系发育关系研究

为了探究陕西省榆林地区鸡蛔虫核糖体18S rRNA、ITS-2基因序列特征及其种系发育关系,试验以采集于该地区的28条鸡蛔虫为样品,PCR扩增其18S rRNA、ITS-2基因的部分序列,测序后分别对鸡蛔虫18S rRNA、ITS-2基因序列的碱基组成进行分析,计算A、T、G和C的含量,并对这两个基因的遗传多样性进行分析,比较其种内、种间差异,构建种系发育树。结果表明:测序所获得的鸡蛔虫18S rRNA、ITS-2基因序列的长度分别为637,350 bp;其中18S rRNA基因序列的A、T、G和C的含量分别为24.5%~25.2%、26.1%~26.4%、28.3%~28.7%和20.3%~20.9%,A+T含量为50.6%~51.6%,G+C含量为48.6%~49.6%,种内差异为0~1.6%;与猪蛔虫(MF358962)、人蛔虫(LC422643)、犬弓首蛔虫(FJ418788)及狮弓蛔虫(JF837179)种间差异为5.18%~61.38%;ITS-2基因序列中的A、T、G和C的含量分别为28.9%~29.1%、38.0%~38.6%、19.1%~19.7%与12.9%~13.7%,A+T含量为66.9%~67.7%,G+C含量为32.0%~33.4%,种内差异为0~2.3%,与猪蛔虫(MF358962)、人蛔虫(LC422643)、犬弓首蛔虫(FJ418788)及狮弓蛔虫(JF837179)种间差异为55.95%~58.42%。此外,所构建的两个种系发育树具有类似的拓扑结构,均显示来自于陕西省榆林地区的28条鸡蛔虫共同聚为一个分支。说明来自于陕西省榆林地区鸡蛔虫的ITS-2序列呈AT偏好,其18S rRNA、ITS-2基因序列均具有种内差异小而种间差异大的特点,且个体之间存在一定程度的遗传差异,但它们之间的亲缘关系较近。

枝睾阔盘吸虫凉山分离株18S rRNA基因部分序列测定与种系发育分析

为探究山羊源枝睾阔盘吸虫的种内遗传变异和系统发育关系,对采集自四川省凉山州9个样品的18S rRNA基因部分序列进行PCR扩增、序列测定及分析,并构建种系发育树。测序结果显示,所测得的9个山羊源枝睾阔盘吸虫18S rRNA基因目标序列长度均为1758 bp,核苷酸同源性为99.9%~100%,共检测到3个单倍型、5个变异位点;系统发育树显示,山羊源枝睾阔盘吸虫凉山州分离株与瘤牛源枝睾阔盘吸虫孟加拉共和国分离株聚在同一小支上,且与胰阔盘吸虫、腔阔盘吸虫亲缘关系最近。本研究结果为枝睾阔盘吸虫病的诊断、分子流行病学调查和枝睾阔盘吸虫种群遗传的进一步研究奠定了基础。

虾肝肠胞虫感染病理特征、18S rRNA基因序列及系统进化树分析

为调查浙江省部分地区虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)暴发来源及与其他宿主来源的微孢子虫进化关系,本研究利用形态学特征和分子鉴定,尝试对引起浙江多地凡纳对虾生长缓慢综合征的病原进行探究.同时基于18S rRNA基因序列分析该病原与其他地区及其他宿主来源微孢子虫的进化关系.结果显示,所采病虾均为EHP阳性,采样地区EHP 18S rRNA基因具有高度同源性,为98.5%~100%.对国内外EHP基因序列进行同源性分析,其同源性为86.2%~100%,所采EHP与印度EHP聚为一个分支. EHP 18S rRNA基因序列与其他水生生物微孢子虫18S rRNA均处于不同分支,表明18S rRNA可作为EHP的主要鉴别序列.将本研究所采EHP与NCBI中同源性较高的比氏肠胞虫和金头鲷微孢子虫比较分析,结果表明微孢子虫基因序列同源性与宿主进化发育程度有关,宿主之间进化距离越近,则微孢子虫基因序列同源性相聚越近.

湖南省怀化地区白纹伊蚊核糖体18S rRNA和线粒体cox 1基因的序列测定及分析

为探究湖南省怀化地区白纹伊蚊的基因变异、遗传多样性及其遗传进化关系,本试验以收集于怀化地区的30只白纹伊蚊为研究对象,利用PCR技术扩增其核糖体18S rRNA及细胞色素c氧化酶亚基Ⅰ(cox1)基因序列,并对其基因变异、遗传多样性及遗传进化关系进行分析。结果显示,所获全部样品的18S rRNA和cox 1基因序列长度均为489和1004 bp;基于18S rRNA基因序列,怀化地区白纹伊蚊的种内差异为0~2.9%,种间差异为28.74%~61.46%;基于cox 1基因序列,怀化地区白纹伊蚊的种内差异为0~0.2%,种间差异为6.08%~12.65%。基于18S rRNA、cox 1基因序列所构建的进化树发现,来自于怀化市的白纹伊蚊全部位于同一分支上。结果表明,怀化地区白纹伊蚊之间存在一定程度的基因变异和遗传多样性,但它们之间的亲缘关系较近,且其18S rRNA、cox 1基因序列的种内差异小、种间差异大,可作为白纹伊蚊鉴定的理想遗传标记。

Comparative evaluation of microscopy,OptiMAL® and 18S rRNA gene based multiplex PCR for detection of Plasmodium falciparum & Plasmodium vivax from field isolates of Bikaner,India

Objective:To evaluate microscopy,OptiMAL? and multiplex PCR for the identification of Plasmodium falciparum（P.falciparum） and Plasmodium vivax（P.vivax） from the field isolates of Bikaner,Rajasthan（Northwest India）.Methods:In this study,a multiplex PCR（P.falciparum and P.vivax） was further developed with the incorporation of Plasmodium malariae（P.malariae） specific primer and also a positive control.The performance of microscopy,plasmodium lactate dehydrogenase（pLDH） based malaria rapid diagnostic test OptiMAL? and 18S rRNA gene based multiplex PCR for the diagnosis of P.falciparum and P.vivax was compared.Results:The three species multiplex PCR if.falciparum,P.vivax and P.malariae) with an inbuilt positive control was developed and evaluated.In comparison with multiplex PCR,which showed the sensitivity and specificity of 99.36%（95%CI,98.11%-100.00%） and 100.00%（95%CI,100.00%-100.00%）,the sensitivity and specificity of microscopy was 90.44%（95%CI,88.849-95.04%） and 99.22%（95% CI,97.71%-100.00%）,and OptiMAL? was 93.58%（95%CI,89.75%-97.42%） and 97.69%（95%CI, 95.10%-100.00%）.The efficiencies were 99.65%,95.10%and 95.45%for multiplex PCK.microscopy and OptiMAL?.respectively.Conclusions:Our results raise concerns over the overall sensitivities of microscopy and OptiMAL?,when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites.This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.

福建省蛇舌形虫18S rRNA基因的扩增及虫种鉴定

以我国福建省三明市某蛇场的病蛇为研究对象,对其进行剖检,观察其器官组织病变,并从体腔中分离得到1对寄生虫成虫,经过剖检观察初步怀疑为舌形虫病。提取虫体和肠道结节DNA,利用18S rRNA基因特异性的引物对提取的DNA进行PCR扩增,经琼脂糖凝胶电泳得到目的条带,切胶回收纯化,连接转化培养得到单菌落。对菌落进行PCR扩增测序。结果显示:病蛇肺脏、肠道病变严重有肿大、淤血和大量结节。经测序得到序列总长均为770 bp,与NCBI公布的相关舌形虫18S rRNA基因序列进行比对分析,三明蛇场得到的舌形虫成虫和肠道结节的DNA序列与EU370434.1(Raillietiella sp.1 BMR-2008 strain C129);AY744887.1(Raillietiella sp.MCZ DNA101107)的相似性为99.74%;与KC904945.1(Raillietiella orientalis)的相似性为97.40%;与JX088397.2(Linguatula serrate isolate NoDog1)的相似性为93.51%。表明此次分离的舟山眼镜蛇舌形虫属于Raillietiella属舌形虫。

First molecular identification of Cryptosporidium by 18S rRNA in goats and association with farm management in Terengganu

Objective:To identify the prevalence of Cryptosporidium from goats in three types of farm management systems in Terengganu,Malaysia and to determine the Cryptosporidium species infecting goats by using 18 S r RNA.Methods:A total of 478 fecal samples were randomly collected from goats in three farms;199 samples were collected from intensive farm,179 samples from semi-intensive farm and 100 samples from extensive farm.The samples were processed by using formolether concentration technique and stained by using modified Ziehl–Neelsen.Positive samples were performed by using nested PCR analysis by using 18 S r RNA.Results:Out of 478 goats,207(43.3%) were found to be infected with Cryptosporidium.Goats reared under the intensive farm management system reported the highest prevalence of infection(49.7%),followed by intensive farm management system(41%) and the lowest prevalence was reported in the goats reared under semi-intensive management system(37.4%).Conclusions:The identified species found in goat was Cryptosporidium parvum.Future study on the zoonotic transmission of Cryptosporidium parvum in goats needs to be done in order to find the source of transmission of this parasite.

基于18S rRNA基因的芜湖市疟疾病例溯源研究

目的研究芜湖市疟疾病例疟原虫种系发育和地理聚类。方法采集2018年8月至2019年9月皖南医学院附属弋矶山医院收治的4例疟疾患者血样,提取血样疟原虫DNA,采用巢式PCR技术对其18S rRNA基因进行扩增、克隆测序分析,并与GenBank中疟原虫相应序列进行比较分析,采用MEGA X软件建树,运用分子进化树分析芜湖市疟疾的地理起源。结果 4例患者PCR电泳结果获得片段均为206 bp,经BLAST比对证实均为恶性疟原虫感染。系统进化树显示,4例病例检测基因片段与GenBank中非洲分离株聚集在相同亚支,其余由GenBank上获取的恶性疟原虫序列也都分别按照地理来源聚集在相同的进化亚支内。结论 18S rRNA基因是能够确定疟疾感染来源地的准确、特异性的溯源分子标记物。

云南省蛋鸡源隐孢子虫鉴定及18S rRNA生物信息学分析

为了了解云南省规模化蛋鸡场腹泻病鸡隐孢子虫的感染情况,试验采用卢戈氏碘液和改良抗酸染色镜检、巢式PCR扩增隐孢子虫18S rRNA基因、BLAST程序测序及遗传进化分析等方法对2020年12月份从云南省多个规模化蛋鸡场采集的71份腹泻蛋鸡的粪便样品进行研究。结果表明:经卢戈氏碘液染色后可见呈棕色近似香蕉形的子孢子,经改良抗酸染色后可见呈红色近似球形的卵囊和近似香蕉形的子孢子。巢氏PCR扩增得到大小为821 bp的目的片段,将获得的虫株命名为YX202106虫株。YX202106虫株与GenBank中已公布的火鸡隐孢子虫的同源性最高,为99.14%~99.51%,且在进化树上处于同一个分支,该虫株还与能引起人畜共患的五种主要隐孢子虫——人隐孢子虫、微小隐孢子虫、火鸡隐孢子虫、猫隐孢子虫、犬隐孢子虫在进化树上构成一个大分支,具有重要的公共卫生学意义。说明云南地区蛋鸡群中存在火鸡隐孢子虫感染,应加强蛋鸡隐孢子虫病的防治。

鱼类肌肉组织18S rRNA降解变化与死亡时间的关联性

自我国实施了海洋伏季休渔制度以来,确定市售鱼类的死亡时间成了渔业执法领域的难题。本论文借鉴了法医学研究,探索死亡鱼类肌肉组织中18S核糖体RNA(18s)降解程度来推断死亡时间的可行性。先通过分析在(0~1℃)低温贮存条件下斑马鱼肌肉组织18s的降解动力学,结果表明肌肉组织内18s呈现区域偏好性的时序降解。在此基础上,建立了一种基于逆转录PCR检测多个特定片长18s降解物的技术,推测鱼类死亡时间的方法原型。虽然该方法仍存在局限性,但在1~7 d的死亡后冷藏时间范围内具有可行性。

16S rRNA及18S rRNA同时检测脑脊液中的病原菌

目的 提高脑脊液中病原菌的快速、准确鉴定率。方法 以细菌的保守序列 16SrRNA和真菌的 18SrRNA为通用引物 ,用多重PCR技术对标准菌株及 4 6例中枢神经系统感染患者的脑脊液进行扩增 ,同时与脑脊液的培养结果进行比较。结果 标准菌株金黄色葡萄球菌、大肠埃希菌、脑膜炎奈瑟菌、结核分枝杆菌、化脓性链球菌及白色念球菌均出现特异条带 ,人白细胞DNA为阴性。 4 6份脑脊液标本培养阳性 39例 ,另有 2例PCR阳性而培养阴性 ,后经诱导传代培养证实为L型金黄色葡萄球菌。PCR阳性 4 1例 ,与培养分离鉴定结果符合率为 10 0 %。结论 多重PCR可快速鉴定脑脊液中病原菌 。

湖南省怀化地区山羊胰阔盘吸虫线粒体pcox1基因和核糖体18S rRNA基因序列遗传变异研究

目的了解湖南省怀化地区山羊体内分离的胰阔盘吸虫遗传变异情况。方法应用PCR技术对分离自湖南怀化地区山羊体内的18株胰阔盘吸虫分离株线粒体细胞色素c氧化酶亚基I基因部分序列(pcox1)和核糖体18S rRNA基因进行扩增,对扩增产物进行测序并进行遗传变异和系统发育分析。结果分离自湖南怀化地区山羊体内的18株胰阔盘吸虫分离株pcox1和18S rRNA基因序列长度分别为430 bp和1 857 bp,分别存在14个和35个变异位点,种内遗传差异分别为0~1.4%和0~0.8%;与GenBank数据库中收录的胰阔盘吸虫中国株基因序列同源性最高,分别为99.0%~99.8%和99.5%~99.8%。基于两种基因构建的系统发育树分析结果一致,本研究获得的18株胰阔盘吸虫分离株与GenBank数据库中已知胰阔盘吸虫分离株聚为同一分支,与支睾阔盘吸虫等阔盘属吸虫相隔较近,与其他吸虫所属分支相隔较远。结论湖南省怀化地区山羊源胰阔盘吸虫分离株遗传变异度较低,线粒体pcox1基因和核糖体18S rRNA基因均适合作为羊源胰阔盘吸虫遗传变异研究的分子标记。

Structural diversity of eukaryotic 18S rRNA and its impact on alignment and phylogenetic reconstruction

Ribosomal RNAs are important because they catalyze the synthesis of peptides and proteins.Comparative studies of the secondary structure of 18S rRNA have revealed the basic locations of its many length-conserved and lengthvariable regions.In recent years,many more sequences of 18S rDNA with unusual lengths have been documented in GenBank.These data make it possible to recognize the diversity of the secondary and tertiary structures of 18S rRNAs and to identify the length-conserved parts of 18S rDNAs.The longest 18S rDNA sequences of almost every known eukaryotic phylum were included in this study.We illustrated the bioinformatics-based structure to show that,the regions that are more length-variable,regions that are less length-variable,the splicing sites for introns,and the sites of A-minor interactions are mostly distributed in different parts of the 18S rRNA.Additionally,this study revealed that some length-variable regions or insertion positions could be quite close to the functional part of the 18S rRNA of Foraminifera organisms.The tertiary structure as well as the secondary structure of 18S rRNA can be more diverse than what was previously supposed.Besides revealing how this interesting gene evolves,it can help to remove ambiguity from the alignment of eukaryotic 18S rDNAs and to improve the performance of 18S rDNA in phylogenetic reconstruction.Six nucleotides shared by Archaea and Eukaryota but rarely by Bacteria are also reported here for the first time,which might further support the supposed origin of eukaryote from archaeans.

基于COⅠ与18S rRNA基因序列的4种肉食螨DNA条形码研究

目的基于线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)与核糖体18S小亚基(18S rRNA)基因序列,分析确定4种肉食螨合适的DNA条形码,从而进一步完善肉食螨DNA条形码数据库。方法2018年5月—2019年7月于安徽省阜阳、芜湖、铜陵等3市小型面粉作坊采集分离肉食螨样本,经形态学鉴定后,提取单个螨基因组DNA,经PCR扩增、克隆和测序获得COⅠ与18S rRNA基因序列,所获序列进行BLAST比对。结合已报道肉食螨序列,用Clustal X 1.83软件进行多序列比对,基于MEGA X软件进行序列分析并计算遗传距离,采用最大似然法构建系统发育树。结果马六甲肉食螨、转开肉食螨、鳞翅触足螨分子鉴定结果与形态学一致,而Gen Bank中缺少网真扇毛螨相关序列。4种肉食螨COI与18S r RNA基因序列(A+T)分别为69.6%和55.1%,碱基替换数分别为137对和46对。基于COⅠ与18S rRNA基因序列变异位点分析,发现4种肉食螨种间变异位点分别为154~321个和58~99个。4种肉食螨COⅠ与18S r RNA基因序列种内遗传距离均≤0.020,种间遗传距离分别为0.235~0.583和0.078~0.114。基于COⅠ与18S rRNA基因序列的系统发育树显示,4种肉食螨均能各自聚为一支,支持率均达100%,与形态学鉴定结果一致。结论线粒体COⅠ基因序列作为4种肉食螨DNA条形码优于18S rRNA基因序列,更适用于探索肉食螨的属、种低阶元亲缘关系。

基于COⅠ、16S rRNA &18S rRNA基因的蚱亚科部分种类分子系统发育分析

本研究扩增和测定了蚱亚科9属30种昆虫的COⅠ、16S rRNA和18S rRNA基因,对联合序列的碱基组成和种间遗传距离进行了分析,同时采用最大似然法(maximum likelihood method, ML)和贝叶斯法(bayesian inference method, BI)构建了分子系统发育关系。结果表明:(1)从遗传距离分析上看,30种蚱类的种间遗传距离范围很小,在0至0.134之间。其中粗股蚱(Tetrix grossifemura)和断隆蚱(Tetrix interrupta)的遗传距离为0,齿股版纳蚱(Bannatettix serrifemoralis)和拟齿股突眼蚱(Ergatettix serrifemoroides)的遗传距离为0.001,它们是同物异名的物种。此外,拟齿股突眼蚱(Ergatettix serrifemoroides)和粗股蚱(Tetrix grossifemura)、拟齿股突眼蚱(Ergatettix serrifemoroides)和断隆蚱(Tetrix interrupta)的遗传距离均为0.005;齿股版纳蚱(Bannatettix serrifemoralis)和粗股蚱(Tetrix grossifemur),齿股版纳蚱(Bannatettix serrifemoralis)和断隆蚱(Tetrix interrupta),短背尖顶蚱(Teredorus brachinota)和武夷山尖顶蚱(Teredorus wuyishanensis),它们之间的遗传距离均为0.006,这些种之间同样存在同物异名的可能。(2)从分子系统发育水平上,仅支持尖顶蚱属(Teredorus)为单系群,不支持悠背蚱属(Euparatettix)、蚱属(Tetrix)、突眼蚱属(Ergatettix)、版纳蚱属(Bannatettix)、柯蚱属(Coptotettix)、庭蚱属(Hedotettix)、台蚱属(Formosatettix)和微翅蚱属(Alulatettix)现行的分类系统。另外,本文从形态分类学的角度分析了分子系统发育水平上不支持蚱亚科现行的分类系统的主要原因。本研究结果将为蚱总科昆虫进行深入的分类修订研究提供科学参考。

Mettl5 mediated 18S rRNA N6-methyladenosine(m^(6)A)modification controls stem cell fate determination and neural function

Ribosome RNA(rRNA)accounts for more than 80%of the cell's total RNA,while the physiological functions of rRNA modifications are poorly understood.Mutations of 18S rRNA m6A methyltransferase METTL5 cause intellectual disability,microcephaly,and facial dysmorphisms in patients,however,little is known about the underlying mechanisms.In this study,we identified METTL5 protein complex and revealed that METTL5 mainly interacts with RNA binding proteins and ribosome proteins.Functionally,we found that Mettl5 knockout in mESCs leads to the abnormal craniofacial and nervous development.Moreover,using Mettl5 knockout mouse model,we further demonstrated that Mettl5 knockout mice exhibit intellectual disability,recapitulating the human phenotype.Mechanistically,we found that Mettl5 maintains brain function and intelligence by regulating the myelination process.Our study uncovered the causal correlation between mis-regulated 18S rRNA m6A modification and neural function defects,supporting the important physiological functions of rRNA modifications in human diseases.

Evaluation of different primers of the 18S rRNA gene to profile amoeba communities in environmental samples

Amoeboid protists,an assemblage of organisms belonging to different phylogenetic lineages,have drawn increasing attention due to their crucial ecological roles in various environments and their potential health risks.Currently,18S rRNA gene sequencing is widely applied for the detection of amoebae.However,it is not clear which is the best primer pair for 18S rRNA gene amplification in amoebae.This study compared the four most commonly used primer pairs for revealing the diversity,composition,core species,and community assembly processes of amoebae in water and sediments.We found that the choice of primers artificially influences the detection of community composition of amoebae.We also found that short-read fragments may lead to mismatches in taxonomy and were not suitable for phylogenetic analyses.In contrast,full-length primers could detect the highest number of amoeba lineages and annotate 80%of reads belonging to amoebae to known species.However,full-length primers did not detect as many amoeba species as V4 primers.Moreover,we showed that beta diversity and community assembly determination were largely unaffected by primer choice,but different primers could influence our interpretations of the ecological process underlying stochasticity and determinism.This study indicates that full-length read sequencing and V4 region Illumina sequencing are suitable for profiling amoeba diversity in the environment.

Molecular systematics analysis of Lymantria dispar based on 18S rRNA and cox1 mtDNA sequence data

The complete sequence of the 18S ribosomal RNA gene （18S rRNA） from Lymantria dispar was cloned and analysed here. 18S rRNA and mitochondrial cytochrome c oxidasel （coxl） gene sequences of Lymantria dispar were compared with homologous sequences of other nine insects from different orders. Analytic results showed that 18S rRNA of these insects had two conserved domains and the second domain was an even more conserved region. The phylogenetic trees based on the full-length sequence and the second domain fragment of 18S rRNA as well as sequence of coxl from different orders indicated that Lepidoptera and Trichoptera, which belongs to Amphiesmenoptera, had a closer phylogenetic relationship and fewer differences were observed comparing with traditional taxonomic results.

Degradation ofβ‑Actin mRNA and 18S rRNA in Mouse Spleen Cells after Death

We observed degradation ofβ‑actin mRNA and 18S rRNA in mouse spleen cells under constant temperature conditions in the different temperature group during postmortem intervals(PMIs)of 0-72 h.Thirty‑nine mice were sacrificed by cervical dislocation and kept at constant temperatures of 10℃,15℃,20℃,25℃,and 30℃.From 0 to 72 h after death,total RNA in spleen cells was extracted every 6 h.The cycle threshold(Ct)values ofβ‑actin mRNA and 18S rRNA were obtained by real‑time‑quantitative polymerase chain reaction.The results showed that,under the conditions of different and constant temperatures after mouse death at 72 h,the Ct values ofβ‑actin and 18S,Ct ratios ofβ‑actin to 18S,and relative ratios ofβ‑actin to 18S were significantly correlated with PMI.In addition,the relative degradation rates ofβ‑actin and 18S appeared to change from fast to slow with the increase of temperature.By interpolation and fitting analysis of the data,we obtained a ternary quintic equation of the relationship between the change in the relative ratios and PMI,which can be used to infer PMI within a certain temperature range(10℃-30℃).