WO2018059195A1 - 一种快速鉴别猪流行性腹泻病毒经典株与变异株的hrm检测引物、试剂盒及方法 - Google Patents

一种快速鉴别猪流行性腹泻病毒经典株与变异株的hrm检测引物、试剂盒及方法 Download PDF

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WO2018059195A1
WO2018059195A1 PCT/CN2017/100435 CN2017100435W WO2018059195A1 WO 2018059195 A1 WO2018059195 A1 WO 2018059195A1 CN 2017100435 W CN2017100435 W CN 2017100435W WO 2018059195 A1 WO2018059195 A1 WO 2018059195A1
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hrm
epidemic diarrhea
porcine epidemic
diarrhea virus
strain
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潘玉
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广州市维佰生物科技有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/701—Specific hybridization probes

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  • the invention belongs to the field of virus detection, and particularly relates to an HRM detection primer, a kit and a method for rapidly identifying a classic strain and a mutant strain of a porcine epidemic diarrhea virus.
  • Porcine Epidemic Diarrhea Virus is a single-stranded positive-strand RNA virus belonging to the Coronavirus family and is the cause of epidemic diarrhea (PED) in pigs of various ages.
  • PED epidemic diarrhea
  • the clinical features of PED are vomiting, diarrhea and dehydration in sick pigs, which are harmful to piglets within one week of age, which can lead to 100% morbidity and 50% to 100% mortality.
  • PED is currently prevalent in many countries including China. It has caused huge losses to the world pig industry. The disease was first transmitted in the UK in 1971. In the 1880s, Asia also reported the occurrence of PED and continued to be popular in Asia.
  • the laboratory diagnostic methods of PEDV mainly include: virus isolation and identification, immunoelectron microscopy, immunofluorescence, virus neutralization test, RT-PCR method, ELISA method and the like.
  • the current diagnostic method can only determine whether the pathogen is PEDV, and it is impossible to distinguish whether the PEDV in the diagnostic sample is a classic strain or a mutant strain.
  • the traditional method for identifying PEDV classical strains and mutant strains is to construct a phylogenetic tree by nucleic acid sequencing, which is time consuming and laborious. Therefore, there is an urgent need for a rapid detection method that can detect both PDEV and whether it is a classic strain or a mutant strain.
  • Another object of the present invention is to provide an HRM detection kit for rapidly identifying classical and variant strains of porcine epidemic diarrhea virus.
  • a further object of the present invention is to provide a method for rapidly identifying HRM detection of classical and variant strains of porcine epidemic diarrhea virus.
  • a HRM primer for rapid identification of classical and variant strains of porcine epidemic diarrhea virus the nucleotide sequence of which is as follows:
  • Primer P1 CGGTTCTTTTCAAAATTTAATG (SEQ ID NO: 1);
  • Primer P2 ATACCATGAACGCCACTA (SEQ ID NO: 2).
  • An HRM detection kit for rapidly identifying classical strains and mutant strains of porcine epidemic diarrhea virus the kit containing the primers described above.
  • a method for rapidly identifying HRM detection of classical and variant strains of porcine epidemic diarrhea virus comprising the following steps:
  • reaction system of RT-PCR amplification in step 2) is:
  • the RT-PCR amplification reaction procedure in the step 2) is as follows: pre-denaturation at 94 ° C for 3 min; denaturation at 94 ° C for 30 s, annealing at 55 ° C for 30 s, extension at 72 ° C for 35 s; 35 cycles; and 72 ° C for 5 min.
  • step 3 the melting curve analysis is performed at a temperature increase rate of 0.2 ° C / step from 80 ° C to 90 ° C.
  • the specific analysis process of the HRM analysis in the step 3) is: in the melting temperature range of 80 to 90 ° C, if the melting curve of the sample to be tested shows a melting peak at 83 to 83.5 ° C, the sample is determined to be a porcine epidemic diarrhea virus.
  • Classical strain if the melting curve of the sample to be tested shows a melting peak at 84.25 to 84.75 ° C, it is determined to be a porcine epidemic diarrhea virus variant strain.
  • the present invention establishes a HRM detection method and primer for rapidly identifying classical strains and mutant strains of porcine epidemic diarrhea virus, and only needs to add a fluorescent saturated dye to a conventional PCR reaction in a PCR system; the detection speed is fast, and all the operation processes are only It takes 2 hours; the cost is low, no specific probe is needed, the amount of fluorescent saturated dye is small, and the cost of saturated dye of 1 sample is about 1 yuan; high-throughput analysis can be realized simply and quickly, suitable for pig epidemic diarrhea Virus screening.
  • the PCR-HRM primer of the present invention has good amplifying property to the classical strain and the mutant strain of porcine epidemic diarrhea virus, and has high PCR amplification efficiency and high detection sensitivity.
  • the PCR-HRM primer of the present invention has good specificity and can specifically amplify porcine epidemic diarrhea virus RNA without amplifying other viral and bacterial pathogens common in pigs, thereby ensuring the reliability of the method.
  • Figure 1 is a HRM normalized melting curve of a standard sample of a classical strain of porcine epidemic diarrhea virus and a mutant strain;
  • Figure 2 is a peak melting curve of HRM for the classical and variant strains of porcine epidemic diarrhea virus
  • Figure 3 is a peak melting curve of HRM for clinical samples of porcine epidemic diarrhea virus classic strains and mutant strains;
  • Figure 4 is a gel diagram of a specific test gel
  • Figure 5 is a peak-shaped melting curve of a classical strain positive plasmid sample
  • Figure 6 is a peak melting curve of a mutant positive plasmid sample
  • Primer P1 CGGTTCTTTTCAAAATTTAATG (SEQ ID NO: 1);
  • Primer P2 ATACCATGAACGCCACTA (SEQ ID NO: 2).
  • the porcine epidemic diarrhea virus RNA in the sample is extracted using the Tiangen RNA extraction kit, and the sample may be intestinal, intestinal contents, feces, vaccine or cell culture.
  • the present invention needs to preferentially prepare a positive standard sample of the classical porcine epidemic diarrhea virus and the mutant strain.
  • Plasmid DNA which was determined to be a classical strain and a mutant strain by sequencing (diluted to a concentration of 10 6 copies/ ⁇ L) was used as a positive standard sample.
  • the positive plasmid samples of the classical and mutant strains of porcine epidemic diarrhea virus obtained above were used as templates, and the upstream and downstream primers of P1 and P2 were subjected to RT-PCR amplification with fluorescent saturation dye, and the preamplification reaction was carried out.
  • the system is:
  • the RT-PCR amplification reaction procedure was: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 35 s; 35 cycles; 72 °C final extension for 5 min; HRM analysis instrument melting temperature setting was Fluorescence signal collection was carried out at a rate of 0.2 ° C from 80 ° C to 90 ° C.
  • the HRM analysis process is performed on a Rotor-Gene Q analyzer that performs the entire PCR-HRM analysis process.
  • PCR amplification can also be performed on a common PCR machine and the PCR product can be directly transferred to the Rotor-Gene Q for melting analysis. Since the lid is not required to be opened, the PCR product is protected from contamination.
  • the HRM results of the standard strains of classical strains and mutant strains of porcine epidemic diarrhea virus are shown in Fig. 1 and Fig. 2.
  • Figure 1 shows the HRM normalized melting curve of the PCR amplification products of the classical strain of porcine epidemic diarrhea virus and the positive plasmid of the mutant strain. It can be seen that the melting curves of the classical strain and the mutant strain are significantly different.
  • Figure 2 shows HRM peaking of PCR amplification products of classical strains of porcine epidemic diarrhea virus and mutant positive plasmid standards.
  • the melting curve in which the melting curve of the classical strain amplification shows a melting peak at 83-83.5 °C, and the melting curve of the mutant strain exhibits a melting peak at 84.25-84.75 °C, the melting peaks of the two are clearly distinguished.
  • RT-PCR amplification is performed using the extracted RNA as a template, and the amplification reaction system is:
  • the PCR amplification reaction procedure was: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 35 s; 35 cycles; 72 °C final extension for 5 min; HRM analysis instrument melting temperature was set at each step. Fluorescence signal collection was performed at a rate of 0.2 ° C from 80 ° C to 90 ° C.
  • the invention has tested 18 clinical samples and vaccine strains, and the test results are shown in Fig. 3.
  • Fig. 3 is a peak-type melting curve, and the melting peaks of the PCR products of the two strains are significantly different.
  • 7 of the samples showed a melting peak at 83-83.5 °C, which was judged to be the classic strain of porcine epidemic diarrhea virus; the other 11 samples had a melting peak at 84.25-84.75 °C, which was judged as porcine epidemic diarrhea virus.
  • Mutant strain 7 of the samples showed a melting peak at 83-83.5 °C, which was judged to be the classic strain of porcine epidemic diarrhea virus; the other 11 samples had a melting peak at 84.25-84.75 °C, which was judged as porcine epidemic diarrhea virus. Mutant strain.
  • Tm melting curve
  • the primer and method of the present invention in the 99% confidence interval and in the melting temperature range of 80-90 ° C, if the melting curve of the sample to be tested shows a melting peak at 83-83.5 ° C, the sample is determined to be a porcine epidemic diarrhea virus.
  • Classical strain if the melting curve of the sample to be tested shows a melting peak at 84.25-84.75 ° C, it is judged to be a porcine epidemic diarrhea virus variant strain. .
  • RNA and DNA viruses susceptible to pigs were swine fever virus (CSFV), porcine transmissible gastroenteritis virus (TGEV), and delta coronavirus (porcine coronavirus).
  • CSFV swine fever virus
  • TGEV porcine transmissible gastroenteritis virus
  • Porcine coronavirus delta coronavirus
  • RV Porcine Circovirus
  • PRV Porcine Pseudorabies Virus
  • PRV Porcine Parvovirus
  • PRRSV Porcine Reproductive and Respiratory Syndrome
  • FMDV Foot and Mouth Disease Virus
  • FIG. 4 shows that in addition to the positive standard control, the target bands were not present in the electropherograms of other non-porcine epidemic diarrhea virus pathogens.
  • the experimental basis for the failure to amplify the non-porcine epidemic diarrhea virus pathogen ensures the specificity of the primers involved in the present invention, and further ensures the reliability of the method of the present invention.
  • Example 2 Using the porcine epidemic diarrhea virus classic strain and the mutant strain positive plasmid sample constructed in Example 2, the sensitivity test was carried out, and the positive plasmid was diluted with the sterilized double distilled water to 10 -9 to a total of 9 dilutions. Sterilized double distilled water was used as a negative control. Amplification reaction of PCR-HRM: The method is the same as that of the reaction system and reaction procedure of Example 2.
  • Figure 5 and Figure 6 show the peak melting curves of the classic and mutant positive plasmid samples, respectively. It can be seen from the figure that the classical plasmid positive plasmids have a specific melting from 10 -1 to 10 -8 . Peak and mutant positive plasmids showed specific melting peaks from 10 -1 to 10 -9 . The results showed that the sensitivity of the method was high, and the detection limits of the positive strains of the classical strain and the mutant strain were 1.35 ⁇ 10 3 copies/ ⁇ L and 192 copies/ ⁇ L, respectively.

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Abstract

一种鉴别猪流行性腹泻病毒经典株与变异株的HRM检测引物、试剂盒及方法。该方法以从样品中提取的病毒核酸为模板，利用所述引物以及荧光饱和染料，进行RT-PCR扩增反应获得扩增产物，对扩增产物进行HRM分析，确定病毒类型。

Description

一种快速鉴别猪流行性腹泻病毒经典株与变异株的HRM检测引物、试剂盒及方法 技术领域
本发明属于病毒检测领域，具体涉及一种快速鉴别猪流行性腹泻病毒经典株与变异株的HRM检测引物、试剂盒及方法。
背景技术
猪流行性腹泻病毒(Porcine Epidemic Diarrhea Virus，PEDV)为单股正链RNA病毒属于冠状病毒科的，是引起各种年龄猪发生流行性腹泻(Porcine Epidemic Diarrhea，PED)的病原。PED临床特征为发病猪呕吐、腹泻、脱水，对一周龄以内仔猪的危害较大，可导致100％发病率及50％～100％致死率，PED目前在包括中国在内的全球多个国家流行，给世界养猪业造成了巨大损失。该疾病于1971年首次在英国被发，19世纪80年代，亚洲也报道了PED的发生，并在亚洲持续流行。随后由于PED弱毒疫苗和灭活疫苗的广泛使用，PED的流行得到一定的控制。但自2010年起，中国主要养猪省份陆续暴发了PED。此次流行的PED主要侵害1周龄内仔猪，造成感染仔猪呕吐、腹泻、继而脱水和急性死亡。随后日本、韩国、泰国等亚洲国家和德国、法国、瑞士、匈牙利及意大利等欧洲国家也相继报道PED的发生。2013年4月，美国爱荷华州首次暴发了PED，而在之前美国并没有发生过PED。自暴发后，很快传遍美国各地，造成了仔猪的高死亡率及大量的经济损失，2014年7月，PED已经在美国31
个州和加拿大部分地区暴发。PEDV CV777的基因组全序列是在2001年确定的，十年后，中国韩国相继公布了许多PEDV全基因组序列。不同PEDV基因组全序列比对结果发现自2010
年以后的流行毒株多个基因发生变异，表明此次世界范围内流行的PED是由PEDV变异株引起的。
由于能够引起腹泻的疾病有很多，临床上很难直接诊断PED。容易造成误诊而造成防疫不当。因此需要结合实验室检测，才能判定是哪种病毒的侵染。现PEDV的实验室诊断方法主要包括：病毒分离与鉴定、免疫电镜、免疫荧光、病毒的中和试验、RT-PCR方法、ELISA方法等。但目前的诊断方法只能判断出病原是否为PEDV，而无法鉴别诊断样本中的PEDV是经典毒株还是变异株。传统的鉴别PEDV经典毒株和变异株的方法是通过核酸测序，构建进化树，该方法费时费力。所以迫切需要一种既可检测PDEV又能够鉴别是经典株还是变异株的快速检测方法。
发明内容
本发明的目的在于提供一种快速鉴别猪流行性腹泻病毒经典株与变异株的HRM检测引物。
本发明的另一目的在于提供一种快速鉴别猪流行性腹泻病毒经典株与变异株的HRM检测试剂盒。
本发明的再一目的在于提供一种快速鉴别猪流行性腹泻病毒经典株与变异株的HRM检测方法。
本发明所采取的技术方案是：
一种快速鉴别猪流行性腹泻病毒经典株与变异株的HRM引物，其核苷酸序列如下所示：
引物P1:CGGTTCTTTTCAAAATTTAATG(SEQ ID NO：1)；
引物P2:ATACCATGAACGCCACTA(SEQ ID NO：2)。
一种快速鉴别猪流行性腹泻病毒经典株与变异株的HRM检测试剂盒，该试剂盒含有上述所述的引物。
一种快速鉴别猪流行性腹泻病毒经典株与变异株的HRM检测方法，包括以下步骤：
1)从样品中提取病毒核酸；
2)以核酸为模板，利用权利要求1所述的引物对P1和P2以及荧光饱和染料，进行RT-PCR扩增反应获得扩增产物；
3)对扩增产物进行HRM分析，确定病毒类型；
上述方法用于非疾病的诊断和治疗。
进一步的，步骤2)中RT-PCR扩增的反应体系为：
5×buffer            4μL
dNTP                 0.8μL
引物P1               0.5μL
引物P2               0.5μL
酶                   0.8μL
模板                 1μL
LC green染料         1μL
ddH2O                11.4μL
Total                20μL。
进一步的，步骤2)中RT-PCR扩增反应程序如下：94℃预变性3min；94℃变性30s，55℃退火30s，72℃延伸35s；循环35次；72℃终延伸5min。
进一步的，步骤3)HRM分析过程中，80℃到90℃以每0.2℃/步的升温速率进行熔解曲线分析。
进一步的，步骤3)中HRM分析的具体分析过程为：在80～90℃熔解温度范围内，若待检测样品熔解曲线在83～83.5℃出现熔解峰时，判定该样品为猪流行性腹泻病毒经典株；若待检测样品熔解曲线在84.25～84.75℃出现熔解峰时，判定为猪流行性腹泻病毒变异株。
本发明的有益效果是：
1)本发明首次建立了一种快速鉴别猪流行性腹泻病毒经典株与变异株的HRM检测方法及引物，只需PCR体系中加入荧光饱和染料进行常规PCR反应；检测速度快，全部操作过程只需2小时；费用低，不需要特异性探针，荧光饱和染料用量少，1个样品的饱和染料成本为1元左右；可以简单、快速的实现高通量分析，适合于猪流行性腹泻病毒筛查。
2)本发明的PCR-HRM引物，对猪流行性腹泻病毒经典株与变异株均有很好的扩增性，PCR扩增效率高，检测灵敏度高。
3)本发明的PCR-HRM引物特异性好，能够特异性扩增猪流行性腹泻病毒RNA而不扩增猪常见的其他病毒性和细菌病原，保证了本方法的可靠性。
附图说明
图1为猪流行性腹泻病毒经典株与变异株标准样品HRM标准化熔解曲线；
图2为猪流行性腹泻病毒经典株与变异株标准样品HRM峰型化熔解曲线；
图3为猪流行性腹泻病毒经典株与变异株临床样品HRM峰型化熔解曲线；
图4为特异性试验凝胶电泳图；
图5为经典株阳性质粒样品的峰型化熔解曲线图；
图6为变异株阳性质粒样品的峰型化熔解曲线图；
具体实施方式
下面结合具体实施例对本发明作进一步的说明，但并不局限于此。
实施例1 HRM引物
本发明经过对所设计的大量引物进行筛选后，发现引物对P1和P2区分猪流行性腹泻病毒经典株与变异株的效果最好，其碱基序列如下所示。
引物P1：CGGTTCTTTTCAAAATTTAATG(SEQ ID NO：1)；
引物P2：ATACCATGAACGCCACTA(SEQ ID NO：2)。
实施例2 标准样品的制备及其PCR-HRM分析
1)猪流行性腹泻病毒RNA的提取：
采用天根RNA提取试剂盒提取样品中的猪流行性腹泻病毒RNA，样品可以是肠道、肠道内容物、粪便、疫苗或细胞培养物。
2)阳性标准样品的制备
为了验证本发明方法可行性与可靠性，同时构建标准阳性样品，为之后的临床样品检测提供HRM阳性对照，本发明需优先制备猪流行性腹泻病毒经典株与变异株阳性标准样品。
分别取经过测序确定为经典株和变异株的质粒DNA(质粒浓度稀释至106copies/μL)，作为阳性标准样品。
3)阳性标准样品的PCR-HRM操作
分别以上述获得的猪流行性腹泻病毒经典株和变异株的阳性质粒样品作为模板，以P1和P2为上下游引物在加有荧光饱和染料下的进行RT-PCR扩增，其预扩增反应体系为：
5×buffer           4μL
dNTP                0.8μL
引物P1              0.5μL
引物P2              0.5μL
酶                  0.8μL
模板                1μL
LC green染料        1μL
ddH2O               11.4μL
Total               20μL。
RT-PCR扩增反应程序为：94℃预变性3min；94℃变性30s，55℃退火30s，72℃延伸35s；循环35次；72℃终延伸5min；HRM分析仪器熔解温度设定是以每步升温0.2℃的速率从80℃到90℃进行荧光信号的收集。
4)阳性标准样品PCR-HRM结果分析
HRM分析过程在Rotor-Gene Q分析仪进行，该仪器可以完成PCR-HRM分析全过程，也可以在普通PCR仪上完成PCR扩增再将PCR产物直接转移至Rotor-Gene Q中完成熔解分析。由于无需开盖，从而保证了PCR产物不受污染。猪流行性腹泻病毒经典株与变异株标准样品HRM结果如图1、图2所示。
图1为猪流行性腹泻病毒经典株与变异株阳性质粒标准品PCR扩增产物的HRM标准化熔解曲线图，可见经典株与变异株熔解曲线差异明显。
图2为猪流行性腹泻病毒经典株与变异株阳性质粒标准品PCR扩增产物的HRM峰型化 熔解曲线图，其中经典株扩增的熔解曲线在83-83.5℃出现熔解峰，变异株扩增的熔解曲线在84.25～84.75℃出现熔解峰时，二者熔解峰区分明显。
实施例3 临床样品的PCR-HRM检测
1)从样本中提取病毒RNA：方法同实施例2中RNA提取方法；
2)以提取的RNA为模板，进行RT-PCR扩增，扩增反应体系为：
5×buffer            4μL
dNTP                 0.8μL
引物P1               0.5μL
引物P2               0.5μL
酶                   0.8μL
模板                 1μL
LC green染料         1μL
ddH2O                11.4μL
Total                20μL
PCR扩增反应程序为：94℃预变性3min；94℃变性30s，55℃退火30s，72℃延伸35s；循环35次；72℃终延伸5min；HRM分析仪器熔解温度设定是以每步升温0.2℃的速率从80℃到90℃进行荧光信号的收集。
3)对扩增产物进行HRM分析，确定毒株类型
本发明对18份临床样本和疫苗株进行了检测，检测结果如图3。
图3为峰型熔解曲线图，两种毒株PCR扩增产物熔解峰值差异明显。通过对图形的分析，发现其中有7份样本出现熔解峰在83-83.5℃，判定为猪流行性腹泻病毒经典株；另外11份样本熔解峰在84.25-84.75℃，判断为猪流行性腹泻病毒变异株。
由于实际检测中熔解曲线Tm受多方面因素的影响会有些变化，包括核酸片段本身、反应试剂盐离子浓度及饱和荧光染料浓度微弱变化，故介定一个宽的Tm范围。
以本发明引物和方法，在99％置信区间内，且在80-90℃熔解温度范围内，若待检测样品熔解曲线在83-83.5℃出现熔解峰时，判定该样品为猪流行性腹泻病毒经典株；若待检测样品熔解曲线在84.25-84.75℃出现熔解峰时，判定为猪流行性腹泻病毒变异株。。
另外，对这18份样品用针对保守基因的引物进行扩增并测序，做进化分析，分析结果与本发明方法检测的结果完全一致，说明本发明方法的准确性高，可达100％。
实施例4 特异性试验
选取猪易感的几种RNA和DNA病毒进行特异性检测，选取的病毒有猪瘟病毒(CSFV)、猪传染性胃肠炎病毒(TGEV)、猪德尔塔病毒(Delta coronavirus)猪轮状病毒(RV)、猪圆环病毒(PCV2)、猪伪狂犬病毒(PRV)、猪细小病毒(PRV)、猪繁殖与呼吸障碍综合症(PRRSV)、口蹄疫病毒(FMDV)。将非猪流行性腹泻病毒样本与阳性标准品按照相同的加样体系和PCR反应条件进行反应，分析PCR产物熔解曲线结果。
特异性试验凝胶电泳图结果如图4所示。图4表明除了阳性标准品对照外，其他非猪流行性腹泻病毒病原电泳图中未出现目的条带。非猪流行性腹泻病毒病原的未能扩增的实验依据保证了本发明所涉及的引物的特异性，进一步也保证了本发明方法的可靠性。
实施例5 灵敏性试验
方法：
用实施例2构建好的猪流行性腹泻病毒经典株与变异株阳性质粒样品进行敏感性试验，阳性质粒用灭菌双蒸水作10倍倍比稀释至10-9，共9个稀释度，并用灭菌双蒸水作阴性对照。PCR-HRM的扩增反应：方法同实施例2的反应体系和反应程序。
敏感性试验PCR-HRM结果分析：
PCR扩增产物用Rotor-Gene Q分析仪进行分析。结果如图5和图6。
图5和图6分别是经典株与变异株阳性质粒样品的峰型化熔解曲线图，从图中可以看出，经典株阳性质粒从10-1稀释至10-8均出现了特异性的熔解峰，变异株阳性质粒从10-1稀释至10-9均出现了特异性的熔解峰。结果表明该方法的灵敏度高，对经典株与变异株阳性质粒的最低检测限分别为1.35x103copies/μL和192copies/μL。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (7)

  1. 一种快速鉴别猪流行性腹泻病毒经典株与变异株的HRM引物，其核苷酸序列如下所示：
    引物P1:CGGTTCTTTTCAAAATTTAATG(SEQ ID NO：1)；
    引物P2:ATACCATGAACGCCACTA(SEQ ID NO：2)。
  2. 一种快速鉴别猪流行性腹泻病毒经典株与变异株的HRM检测试剂盒，其特征在于：该试剂盒含有权利要求1所述的引物。
  3. 一种快速鉴别猪流行性腹泻病毒经典株与变异株的HRM检测方法，其特征在于，包括以下步骤：
    1)从样品中提取病毒核酸；
    2)以核酸为模板，利用权利要求1所述的引物对P1和P2以及荧光饱和染料，进行RT-PCR扩增反应获得扩增产物；
    3)对扩增产物进行HRM分析，确定病毒类型；
    上述方法用于非疾病的诊断和治疗。
  4. 根据权利要求3所述的方法，其特征在于，步骤2)中RT-PCR扩增的反应体系为：
    Figure PCTCN2017100435-appb-100001
  5. 根据权利要求3或4所述的方法，其特征在于，步骤2)中RT-PCR扩增反应程序如下：94℃预变性3min；94℃变性30s，55℃退火30s，72℃延伸35s；循环35次；72℃终延伸5min。
  6. 根据权利要求3所述的方法，其特征在于：步骤3)HRM分析过程中，80℃到90℃以每0.2℃/步的升温速率进行熔解曲线分析。
  7. 根据权利要求3所述的方法，其特征在于：步骤3)中HRM分析的具体分析过程为：在80～90℃熔解温度范围内，若待检测样品熔解曲线在83～83.5℃出现熔解峰时，判定该样品为猪流行性腹泻病毒经典株；若待检测样品熔解曲线在84.25～84.75℃出现熔解峰时，判定为猪流行性腹泻病毒变异株。
PCT/CN2017/100435 2016-09-27 2017-09-05 一种快速鉴别猪流行性腹泻病毒经典株与变异株的hrm检测引物、试剂盒及方法 WO2018059195A1 (zh)

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