CN103773858A - 副溶血性弧菌三重实时荧光pcr检测试剂盒 - Google Patents
副溶血性弧菌三重实时荧光pcr检测试剂盒 Download PDFInfo
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Abstract
本发明提供一种副溶血性弧菌三重实时荧光PCR检测试剂盒,包括三对特异性引物、EvaGreenPCR预混液、阴性对照和阳性对照,特异性引物包括副溶血性弧菌耐热直接溶血素基因(tdh)、耐热相关溶血素基因(trh)和毒素表达调控蛋白基因(toxR)的特异性引物。本发明试剂盒不但具有实时荧光PCR快速、灵敏和自动化程度高的优点,而且基于熔解曲线技术进行三重实时荧光PCR检测,不需要合成昂贵的检测探针,检测成本低,只需最为常规的FAM/SYBR通道即可完成3个靶基因的检测,对实时荧光PCR仪的要求低,几乎适用于所有市售实时荧光PCR仪。所述检测周期短、检测成本低、检测结果可靠。
Description
技术领域
本发明属于生物技术领域,涉及副溶血性弧菌三重实时荧光PCR检测试剂盒和检测方法。
背景技术
副溶血性弧菌(Vibrio parahaemolyticus)是1953年由Fujino等从日本一起食物中毒患者体内分离到的一种革兰氏阴性嗜盐弧菌,是弧菌属内比较重要的致病菌之一,进食被该菌污染的食物会引起食物中毒,临床上以急性起病、腹痛、呕吐、腹泻及水样便为主要症状。副溶血性弧菌是一种海洋细菌,主要来源于鱼、虾、蟹、贝类和海藻等海产品。该菌存活能力强,是沿海地区细菌性食物中毒中最为常见的病原菌。副溶血性弧菌传统的检测方法包括前增菌、选择性增菌、选择性培养、生化鉴定和血清学实验等过程,操作繁琐,鉴定周期长。如果要确定菌株是否含有毒力因子,还需增加其他特殊的生化鉴定等实验,鉴定周期更加漫长。近年来随着分子生物学的发展,PCR技术被越来越多的运用到病原菌的鉴定中来。PCR技术包括普通PCR和实时荧光PCR,普通PCR虽然具有操作简单,检测成本低等优点,但是需要对扩增产物进行电泳跑胶等后处理,不易自动化和标准化。实时荧光PCR是利用荧光信号伴随着PCR产物的扩增而增加的原理,在PCR扩增过程中,连续不断地检测反应体系中荧光信号的变化,实时在线监测反应过程,再结合相应的软件对产物进行分析,一次上机即可完成结果检测,易自动化。实时荧光PCR主要分为探针类和染料类两种,探针类实时荧光PCR需要合成荧光标记的探针,由于探针的合成成本较高,所以造成探针类实时荧光PCR检测成本高;染料类实时荧光PCR虽然检测成本低,但是由于早期主要使用非饱和荧光染料SYBR Green I,存在着稳定性差和染料重分布等问题,检测灵敏度低而且不适用于多重实时荧光PCR,限制了染料类实时荧光PCR在检验检测领域的应用,随着饱和荧光染料Eva Green和LC Green等的出现,染料类实时荧光PCR的检测灵敏度显著提高,而且由于解决了染料重分布问题可以结合熔解曲线技术用于多重实时荧光PCR检测,因此已经在检验检测领域显示出了巨大的优势。
发明内容
本发明的目的是提供一种基于熔解曲线技术的副溶血性弧菌三重实时荧光PCR检测试剂盒,通过一次反应快速、经济和可靠的完成对副溶血性弧菌的特异性检测和毒力基因检测。
本发明试剂盒由EvaGreen PCR预混液、三对特异性引物、阳性对照、阴性对照组成,EvaGreen PCR预混液包括PCR缓冲液、dNTP混合物、DNA聚合酶和饱和荧光染料EvaGreen;阳性对照为tdh、trh双阳性的副溶血性弧菌DNA提取液;阴性对照为超纯水。
EvaGreen PCR预混液可购买商品化的EvaGreen PCR预混液,也可自行配制。
三对特异性引物如下:
tdh上游引物:5’-CTTCCATCTGTCCCTTTTCCTGCC-3’
tdh下游引物:5’-CCTGACGTTGTGAATACTGATTGACCATA-3’
trh上游引物:5’-TACCTTTTCCTTCTCCAGGTTCGG-3’
trh下游引物:5’-TCGTTTTATGTTTCGGTTTGTCCAGT-3’
toxR上游引物:5’-TCTTCTGACGCAATCGTTGAACCA-3’
toxR下游引物:5’-CTGATACTCACCAATCTGACGGAACTG-3’。
本发明利用该试剂盒检测方法如下:
(1)提取待测样品DNA;
(2)取特异性引物和EvaGreen PCR预混液,分别加入阴性对照、阳性对照或待测样品DNA形成扩增反应体系;
(3)将扩增反应体系置于实时荧光PCR仪上进行实时荧光PCR反应,反应条件为:95℃预变性2min,95℃5s、59℃25s进行30个循环,在退火阶段采集荧光,最后进行熔解曲线监测,即完成上述最后一个循环后,温度升至75℃,保温5s后以0.1℃/s的升温速率逐渐升至85℃,在此升温过程中进行荧光强度的连续检测。并根据熔解曲线判断样品中是否含有副溶血性弧菌(样品是否为副溶血性弧菌),以及该副溶血性弧菌的毒力基因携带情况,tdh、trh和toxR基因扩增产物的Tm值分别为79.5±0.1℃、77.5±0.1℃和82±0.1℃。
利用三重实时荧光PCR方法,以副溶血性弧菌的toxR基因、tdh基因和trh基因为靶基因。toxR基因是副溶血性弧菌特异性检测中常用的靶基因;tdh和trh基因编码的耐热直接溶血素和耐热相关溶血素具有溶血活性和肠毒素作用,被认为是副溶血性弧菌的主要毒力因子。
本发明的关键在于扩增引物的设计,染料类实时荧光PCR能用于多重检测是因为如果不同的扩增产物长度不同、其GC比值不同,进行熔解曲线分析时就会有其独特的熔解曲线,并且熔解曲线性质、位置和Tm值都是不同的。因此引物设计时不但要采用blast分析保证引物的特异性,还要考虑扩增产物的长度和GC比值,保证进行熔解曲线分析时不同的靶基因的能产生具有不同Tm值的熔解曲线。除引物外本发明所需其他试剂,如EvaGreen PCR预混液,包括PCR缓冲液、dNTP混合物、DNA聚合酶和饱和荧光染料EvaGreen,EvaGreen PCR预混液可购买商品化的EvaGreen PCR预混液,也可自行配制,本发明中使用商品化的Bio-Rad SsoFast EvaGreen预混液;阳性对照为tdh、trh双阳性的副溶血性弧菌DNA提取液;阴性对照为超纯水。
本发明的优势主要体现在:(1)具有实时荧光PCR快速、灵敏和自动化程度高的优点;(2)不需要合成昂贵的检测探针,检测成本低;(3)探针法三重实时荧光PCR需要3个不同的荧光通道才能完成3个靶基因的检测,因此不适用于部分只有两个荧光通道的实时荧光PCR仪,如Bio-Rad MyiQ 2,本发明只需最为常规的FAM/SYBR通道即可完成3个靶基因的检测,几乎适用于所有市售实时荧光PCR仪。
附图说明
图1是不同菌株的实时荧光PCR扩增曲线图。
图2是不同浓度DNA模板的实时荧光PCR扩增曲线图。
图3是不同浓度DNA模板对应的标准曲线。
图4是tdh阳性trh阴性副溶血性弧菌的熔解曲线分析图。
图5是tdh、trh双阳性副溶血性弧菌的熔解曲线分析图。
具体实施方式
本发明结合附图和实施例作进一步的说明。
实施例1:
本发明试剂盒由EvaGreen PCR预混液、三对特异性引物、阳性对照、阴性对照组成,EvaGreen PCR预混液包括PCR缓冲液、dNTP混合物、DNA聚合酶和饱和荧光染料EvaGreen;阳性对照为tdh、trh双阳性的副溶血性弧菌DNA提取液;阴性对照为超纯水。
EvaGreen PCR预混液可购买商品化的EvaGreen PCR预混液,也可自行配制。
三对特异性引物如下:
tdh上游引物:5’-CTTCCATCTGTCCCTTTTCCTGCC-3’
tdh下游引物:5’-CCTGACGTTGTGAATACTGATTGACCATA-3’
trh上游引物:5’-TACCTTTTCCTTCTCCAGGTTCGG-3’
trh下游引物:5’-TCGTTTTATGTTTCGGTTTGTCCAGT-3’
toxR上游引物:5’-TCTTCTGACGCAATCGTTGAACCA-3’
toxR下游引物:5’-CTGATACTCACCAATCTGACGGAACTG-3’。
利用本实施例试剂盒检测方法:
提取已经过鉴定的tdh、trh双阳性的副溶血性弧菌菌株DNA为模板,用三对特异性引物在实时荧光PCR仪上进行扩增反应,20μL扩增反应体系为:模板DNA1μL,Bio-Rad SsoFast EvaGreen预混液10μL,10μmol/L toxR上下游引物各0.22μL,10μmol/L tdh上下游引物各1.6μL,10μmol/L trh上下游引物各0.8μL,超纯水补足至20μL。扩增反应条件为:95℃预变性2min,95℃5s、59℃25s进行30个循环,在退火阶段采集荧光,最后进行熔解曲线监测,即完成上述最后一个循环后,温度升至75℃,保温5s后以0.1℃/s的升温速率逐渐升至85℃,在此升温过程中进行荧光强度的连续检测。并根据熔解曲线判断样品中是否含有副溶血性弧菌(样品是否为副溶血性弧菌),以及该副溶血性弧菌的毒力基因携带情况,tdh、trh和toxR基因扩增产物的Tm值分别为79.5±0.1℃、77.5±0.1℃和82±0.1℃。本发明中使用商品化的Bio-Rad SsoFast EvaGreen预混液。
实施例2:特异性实验
以白色念珠菌(ATCC 10231)、阪崎肠杆菌(ATCC 51329)、大肠埃希氏菌(ATCC 25922)、伤寒沙门菌(ATCC 50097)、金黄色葡萄球菌(ATCC 25923)、单增李斯特菌(CMCC 54006)、福氏志贺氏菌(ATCC 12022)、铜绿假单胞杆菌(ATCC 27853)、小肠结肠炎耶尔森氏菌(ATCC 23715)、蜡样芽孢杆菌(ATCC 11778)、拟态弧菌(ATCC 33653)、创伤弧菌(ATCC 27562)溶藻弧菌(ATCC 17749)和霍乱弧菌作为待测样品,副溶血性弧菌标准菌株(ATCC 33847)和tdh、trh双阳性的副溶血性弧菌菌株作为阳性对照,超纯水作为阴性对照。白色念珠菌(ATCC 10231)、阪崎肠杆菌(ATCC 51329)、大肠埃希氏菌(ATCC 25922)、伤寒沙门菌(ATCC 50097)、金黄色葡萄球菌(ATCC 25923)、福氏志贺氏菌(ATCC 12022)、铜绿假单胞杆菌(ATCC 27853)、小肠结肠炎耶尔森氏菌(ATCC 23715)、蜡样芽孢杆菌(ATCC 11778)、拟态弧菌(ATCC 33653)、创伤弧菌(ATCC 27562)、溶藻弧菌(ATCC 17749)和副溶血性弧菌(ATCC 33847)来源于美国模式培养物集存库;单增李斯特菌(CMCC 54006)来源于中国医学细菌保藏管理中心; tdh、trh双阳性的副溶血性弧菌和霍乱弧菌分离自腹泻病人,均按照国家标准进行了严格的生理生化鉴定,tdh、trh双阳性的副溶血性弧菌还进行了严格的毒力基因鉴定。按照实施例1所述方法进行检测,如图1所示,阳性对照均产生了明显的扩增曲线,其他菌株和阴性对照均无扩增曲线产生。
实施例3:灵敏度实验
以tdh、trh双阳性副溶血性弧菌菌株DNA提取液为模板,用超纯水稀释到50ng/μL、5 ng/μL、500pg/μL、50 pg/μL、5 pg/μL和500 fg/μL等一系列稀释度,每个稀释度各取1μL模板DNA按照实施例1所述方法进行检测,每个稀释度各做3个复孔。结果显示,即使扩增反应体系中仅含有5pg的模板DNA也能产生明显的扩增曲线,而且模板DNA的浓度与Cq值有良好的线性关系,浓度越高Cq值越低,检测结果参见图2,图3。
实施例4:副溶血性弧菌标准菌株检测
以副溶血性弧菌标准菌株ATCC 33847的DNA提取液为模板,按照实施例1所述方法进行检测。结果显示,ATCC 33847为tdh阳性trh阴性副溶血性弧菌,与该菌株的实际情况相符。
实施例5:副溶血性弧菌分离株检测
以腹泻病人中分离到的35株副溶血性弧菌菌株DNA提取液为模板,按照实施例1所述方法进行检测。结果显示,32株副溶血性弧菌菌株为tdh阳性trh阴性菌株,3株副溶血性弧菌菌株为tdh、trh双阳性菌株,与本实验室前期采用其他方法检测的结果相符。
实施例6:食物中毒患者肛拭子样本检测
将某次食物中毒中采集到的肛拭子样本放入副溶血性弧菌增菌液中增菌5小时,取100μL增菌液离心收集菌体,按照实施例1所述方法进行检测。其中12份样本检测结果为副溶血性弧菌阳性,其余样本阴性,12份阳性样本均为tdh阳性trh阴性副溶血性弧菌。分离培养和生化鉴定的结果也显示这12份样本副溶血性弧菌阳性,其余样本阴性,对分离株进一步进行毒力基因鉴定的结果也与本发明的检测结果一致。
<110>嘉兴市疾病预防控制中心
<120>副溶血性弧菌三重实时荧光PCR检测试剂盒
<160>6
<210>1
<211>24
<212>DNA
<213>人工序列
<400>1
CTTCCATCTG TCCCTTTTCC TGCC 24
<210>2
<211>29
<212>DNA
<213>人工序列
<400>2
CCTGACGTTG TGAATACTGA TTGACCATA 29
<210>3
<211>24
<212>DNA
<213>人工序列
<400>3
TACCTTTTCC TTCTCCAGGT TCGG 24
<210>4
<211>26
<212>DNA
<213>人工序列
<400>4
TCGTTTTATG TTTCGGTTTG TCCAGT 26
<210>5
<211>24
<212>DNA
<213>人工序列
<400>5
TCTTCTGACG CAATCGTTGA ACCA 24
<210>6
<211>24
<212>DNA
<213>人工序列
<400>6
CTGATACTCA CCAATCTGAC GGAACTG 27
Claims (2)
1. 一种副溶血性弧菌三重实时荧光PCR检测试剂盒,其特征在于,该试剂盒包含三对特异性引物、EvaGreen PCR预混液、阳性对照和阴性对照,其中EvaGreen PCR预混液包括PCR缓冲液、dNTP混合物、DNA聚合酶和饱和荧光染料EvaGreen,阳性对照为tdh、trh双阳性副溶血性弧菌DNA提取液,阴性对照为超纯水,所述三对特异性引物的序列如下:
tdh上游引物:5’-CTTCCATCTGTCCCTTTTCCTGCC-3’
tdh下游引物:5’-CCTGACGTTGTGAATACTGATTGACCATA-3’
trh上游引物:5’-TACCTTTTCCTTCTCCAGGTTCGG-3’
trh下游引物:5’-TCGTTTTATGTTTCGGTTTGTCCAGT-3’
toxR上游引物:5’-TCTTCTGACGCAATCGTTGAACCA-3’
toxR下游引物:5’-CTGATACTCACCAATCTGACGGAACTG-3’ 。
2.根据权利要求1所述的一种副溶血性弧菌三重实时荧光PCR检测试剂盒,其特征在于,EvaGreen PCR预混液可购买商品化的EvaGreen PCR预混液,也可自行配制。
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CN111041111A (zh) * | 2020-01-08 | 2020-04-21 | 厦门海关技术中心 | 同时检测三种基因型的副溶血性弧菌的三重pcr引物、试剂盒及其检测方法 |
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