WO2018219214A1 - 一种快速鉴别猪繁殖与呼吸综合症病毒疫苗株与其它毒株的检测方法 - Google Patents

一种快速鉴别猪繁殖与呼吸综合症病毒疫苗株与其它毒株的检测方法 Download PDF

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WO2018219214A1
WO2018219214A1 PCT/CN2018/088303 CN2018088303W WO2018219214A1 WO 2018219214 A1 WO2018219214 A1 WO 2018219214A1 CN 2018088303 W CN2018088303 W CN 2018088303W WO 2018219214 A1 WO2018219214 A1 WO 2018219214A1
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probe
pcr
hrm
strain
vaccine strain
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PCT/CN2018/088303
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French (fr)
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郭鹏举
饶丹
丛锋
黄韧
陈梅丽
练月晓
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广东省实验动物监测所
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

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  • the invention belongs to the field of virus detection, and particularly relates to a PCR-HRM detection method for rapidly identifying a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine strain GDr180 strain and other strains.
  • PRRSV porcine reproductive and respiratory syndrome virus
  • PRRS Porcine Reproductive and Respiratory Syndrome
  • the PRRSV attenuated vaccine strain (GDr180) is a newly-produced high-generation attenuated strain of the Chinese Veterinary Drug Control Institute. It has a good immunoprotective and safe vaccine strain. Clinical trials have shown that the vaccine is highly immune to experimental pigs. Protective effects. After vaccination, pigs will not cause immune side effects such as elevated body temperature, and reduce the risk of live vaccine detoxification. Strong immune protection and a longer immune protection period can be obtained after vaccination. The development of GDr180 vaccine enriches the types of highly pathogenic blue ear vaccines in China's market. By identifying vaccine strains and wild strains, it is a very meaningful measure to exclude infections of vaccine strains or wild strains after immunization.
  • PCR-high-resolution melting curve analysis is a simple, fast, and low-cost method for single nucleotide polymorphism (SNP) and genotyping.
  • Post-PCR amplification detection technology for high-throughput mutation scanning and genotyping.
  • the difficulty of the PCR-HRM experiment is that it is necessary to find a suitable target site to design primers.
  • the melting curve probe peak appears to have a difference in melting temperature to achieve the purpose of differentiation, and the melting temperature is The size of the difference depends on the degree of sequence matching and is also related to the type and arrangement of the base sequence. A good target sequence and appropriate PCR amplification conditions can achieve a good melting curve discrimination effect. If not, the melting curve probe peaks that are different can not be obtained, and the purpose of differentiation cannot be achieved.
  • the object of the present invention is to provide a primer and a probe for rapidly identifying a PCR-HRM detection method for a PRRSV vaccine strain GDr180 strain and other strains;
  • Another object of the present invention is to provide a reagent for rapidly identifying a PCR-HRM method for detecting a PRRSV vaccine strain GDr180 strain and other strains;
  • a further object of the present invention is to provide a PCR-HRM detection method for rapidly identifying a PRRSV vaccine strain GDr180 and other strains.
  • a primer for rapid identification of the PCR-HRM assay for the PRRSV vaccine strain GDr180 strain and other strains the nucleotide sequence of which is as follows:
  • a probe for rapidly identifying a PCR-HRM assay for the PRRSV vaccine strain GDr180 strain and other strains the nucleotide sequence of which is as follows:
  • Probe 1 AATGTGTCAGCACTGTGGCTGTGTG.
  • one end of the probe is modified and blocked with a modification comprising an amino-modified C6, a reverse dT, and a C3 spacer (C3 spacer).
  • a modification comprising an amino-modified C6, a reverse dT, and a C3 spacer (C3 spacer).
  • C3 spacer C3 spacer
  • a PCR-HRM detection reagent for rapidly identifying the PRRSV vaccine strain GDr180 and other strains which contains the primers and probes described above.
  • a rapid PCR method for PCR-HRM detection of PRRSV vaccine strain GDr180 and other strains including the following steps:
  • the PCR preamplification reaction system in step 3 is:
  • the PCR pre-amplification reaction procedure in step 3) is: pre-denaturation at 94 ° C for 5 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.
  • the PCR-HRM amplification reaction system in step 4) is:
  • the PCR-HRM amplification reaction procedure in step 4) is: denaturation at 98 ° C for 10 sec, annealing at 55 ° C for 30 sec, extension at 72 ° C for 20 sec, 45 cycles; HRM heating step is 92 ° C for 1 min, 40 ° C for 2 min; 50 ° C The temperature rise rate was 0.5 ° C / step to 90 ° C.
  • the HRM analysis method is as follows:
  • the method of the invention combines PCR and high-resolution melting curve technology to screen the specific base sites of the GDr180 strain, and by adding the oligonucleotide strands blocked at the 3' end, the melting curve is determined according to the difference of the sequence matching degree of the target sites. The difference in melting temperature occurs at the peak to achieve the purpose of differentiation.
  • the inventors finally found a specific gene locus that differs from other vaccine strains and wild strains by GDr180 strain, obtained the strand bound to the probe by asymmetric PCR amplification, and obtained the melting curve by HRM analyzer.
  • the probe peak of the melting curve is analyzed using the derivative value of the fluorescence value.
  • the invention establishes a PCR-HRM differential detection method and primer for rapidly distinguishing PRRSV vaccine strain GDr180 from other strains for the first time, and the operation of PCR combined with HRM analysis is simple, and the whole process takes only 3 hours, which greatly shortens the time required for typing.
  • the cost is low, the required probe is closed at the 3' end of the oligonucleotide C3, the synthesis price is low, and the fluorescent saturated dye is cheap and easy to obtain; because the experimental method has good specificity and good repeatability, it can be accurate, fast and high-throughput. It is used for the screening of PRRSV specific vaccine strains, providing a basis for rapid clinical diagnosis.
  • the PCR-HRM primer of the invention has good amplifying property to GDr180 and other strains, and has high PCR amplification efficiency and high detection sensitivity.
  • the PCR-HRM primer of the present invention has good specificity, can specifically amplify PRRSV, and has no amplification property to other pathogens, and the present invention has reliability for screening specific strains.
  • Figure 1 is a peak melting curve of HRM of PRRSV vaccine strain GDr180 and other strains
  • Figure 2 is a peak melting curve of HRM of clinical samples of PRRSV vaccine strain GDr180 and other strains;
  • Figure 3 is a PCR-HRM primer-specific gel electrophoresis pattern of PRRSV vaccine strain GDr180 and other strains.
  • the method of the invention combines PCR and high-resolution melting curve technology to screen the specific base sites of the GDr180 strain, and by adding the oligonucleotide strands blocked at the 3' end, the melting curve probe is made according to the difference of the matching degree of the target site sequences. Peaks show differences in melting temperatures for differentiation purposes.
  • the inventors selected specific primers and probes by repeated screening, and the nucleotide sequences are as follows:
  • P1 5'-TTCTTCTTGCCTTTTCTATGCTTC-3' (SEQ ID NO: 1);
  • P2 5'-CAACTGTGTCAAGGAAATGGCT-3' (SEQ ID NO: 2);
  • P3 5'-CAGGATAGGGCATGACCGAT-3' (SEQ ID NO: 3);
  • P4 5'-CCAAATATCTCGGGATGGAACT-3' (SEQ ID NO: 4);
  • Probe 1 5'-AATGTGTCAGGCACTGTGGCTGTGTG-C3-3' (SEQ ID NO: 5).
  • One end of the probe Probe 1 was modified and blocked with a modification including an amino-modified C6, a reverse dT, and a C3 spacer (C3 spacer).
  • the modification used in the present invention is C3 spacer, the C3 blocked probe is inexpensive and the synthesis technology is mature, and the fluorescent saturated dye type is selected in a large amount and used in a small amount, which is advantageous for the promotion of the technology of the present invention.
  • the length of the amplified fragment of P1/P2 primer is 525 bp, which can efficiently amplify all PRRSV strains;
  • the length of the amplified fragment of P3/P4 primer is 137 bp, this sequence contains GDr180 strain and other vaccine strains and wild strains.
  • Probe 1 is a target sequence of the P3/P4 amplified region, which is obtained by asymmetric PCR amplification to obtain a chain bound to the probe, and then further obtained by a HRM analyzer to obtain a melting curve, using fluorescence values The values are used to analyze the probe peaks of the melting curve.
  • the mixture was homogenized thoroughly on ice in a tissue homogenizer, repeatedly frozen and thawed three times, centrifuged at 10,000 rpm for 5 min, and the supernatant was taken for use (conditional filtration can be sterilized).
  • the tissue supernatant or serum was subjected to RNA extraction using the Tiangen Trizol reagent (Tiangen Biochemical) instructions.
  • the present invention preferentially prepares PRRSV GDr180 strain and two other non-GDr180 strain standard samples (including HuN4-F112, MLV), using NTC as a negative blank control.
  • plasmid DNA which was determined to be GDr180, HuN4-F112 and MLV strains by sequencing was used as a template (plasmid concentration was diluted to 10 6 copies/ ⁇ L), and P3 and P4 were used as primers for upstream and downstream (plasmid standard) No pre-amplification is required.
  • Probe1 is a blocking probe and is subjected to PCR amplification under the addition of a saturated fluorescent dye.
  • the pre-amplification reaction system is:
  • the results are shown in Figure 1.
  • the asymmetric PCR amplification products of the three standards have clear melting curves.
  • the Tm value of the melting curve probe peak of the GDr180 strain is 72.50 ° C, and the Tm value of the main melting peak (PCR product melting peak) 84.60 ° C; non-GDr180 strain HuN4-F112 probe peak Tm value of 64.3 ° C, the main melting peak Tm value of 84.95 ° C; MLV probe peak Tm value of 65.55 ° C, the main melting peak Tm value of 85.12 ° C.
  • the melting curve of the GDr180 strain can be well distinguished from other standard melting curves by the difference in probe peak Tm.
  • the Tm value of the PCR product melting curve probe peak of different batches of GDr180 vaccine strain was referenced, the probe peak Tm value was 72.64 ⁇ 0.39 °C, and the main peak Tm value was 84.7 ⁇ 0.29 °C.
  • the Tm value of the probe peak is 72.64 ⁇ 1.17°C, and the Tm value of the main peak is 84.7 ⁇ 0.87°C, it is judged as the vaccine strain GDr180; if the probe peak is not in this range or the PRRSV strain without the probe peak Then, it was judged to be other PRRSV strains other than the GDr180 strain.
  • the amplification reaction system is:
  • the melting curve obtained by the vaccine strain GDr180 standard has obvious probe peak and main peak, the probe peak Tm value is 72.68 ° C, the main peak Tm value is 84.8 ° C; the HuN4-F112 probe peak Tm value is 63.9 ° C, the main peak Tm The value was 84.75 ° C, the probe peak of MLV was 65.65 ° C, and the main peak was 85.35 ° C.
  • the probe peak Tm value of 6 of 7 clinical samples was 65.63 ⁇ 0.23 °C (one of the samples had no probe peak, because the probe was too different from the target sequence), and the Tm value of the main peak of the PCR product melting of 7 samples was 85.13 ⁇ 0.25 ° C.
  • the probe peak Tm value was 65.63 ⁇ 0.69°C, and there was no crossover with the GDr180 strain (72.64 ⁇ 1.17°C), and the probe peak Tm value was extremely significant. Therefore, all of the seven isolates were judged to be non-GDr180 strains of PRRSV strain.
  • Sample 4 65.75 85.00 Sample 5 65.6 85.25 Sample 6 65.85 85.50 Sample 7 65.85 85.40
  • the porcine-derived viruses PEDV, PRV, CSFV, PCV and PoRV were selected as negative controls, and P1/P2 primers were selected. The results are shown in Figure 3.
  • lanes 1-5 are PEDV, PRV, CSFV, PCV and PoRV, and gel electrophoresis shows that the target band is not expanded.
  • Lanes 6-7 are PRRSV positive reference GDr180 strain and HuN4-F112, respectively, with clear purpose strips. Band, indicating that the specificity of primer amplification is good.

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Abstract

本发明公开了一种快速鉴别PRRSV疫苗株GDr180株与其他毒株的PCR-HRM检测方法。本发明方法为PCR基础上附带HRM，包括第一轮PCR常规扩增及第二轮PCR-HRM，反应之前加荧光饱和染料。

Description

一种快速鉴别猪繁殖与呼吸综合症病毒疫苗株与其它毒株的检测方法 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繁殖与呼吸综合症病毒(PRRSV)疫苗株GDr180株与其他毒株的PCR-HRM检测方法。
背景技术
猪繁殖与呼吸综合症(Porcine Reproductive and respiratory syndrome，PRRS)最早于1987年发现于美国，其症状主要表现为怀孕母猪流产、早产、产死胎、木乃伊胎及仔猪感染后的成活率下降，成年猪的呼吸道症状。
PRRSV弱毒疫苗株(GDr180)是中国兽医药品监察所最新培育的高代次致弱毒株，具有良好免疫保护力和安全性的疫苗株，临床试验证明该疫苗免疫接种对实验猪具有很好的免疫保护作用。疫苗接种后不会使猪产生体温升高等免疫副反应，降低活疫苗排毒风险。接种后可获得坚强的免疫保护和较长的免疫保护期。GDr180疫苗的研制丰富了我国市场高致病蓝耳疫苗的种类，通过鉴别疫苗株与野毒株可以排除免疫后是疫苗株残毒还是野毒株的感染，是非常有意义的举措。目前较少关于GDr180株与野毒株的鉴别研究，仅有双重荧光定量PCR方法的研究报道，但需要使用较昂贵的MGB荧光探针，成本高。所以，建立一种较简单、经济的鉴别疫苗株与野毒株的方法，具有重大意义。
PCR-高分辨熔解曲线分析技术(high-resolution melting curve analysis，HRM)是一种用于单核苷酸多态性(SNP)和基因分型研究方法，是一种简单、快速、低成本的PCR扩增后检测技术，可用于高通量的突变扫描和基因分型。PCR-HRM实验的难点在于，需要找筛选出合适的靶位点来设计引物，依据靶位点的序列匹配度的差异使得熔解曲线探针峰出现熔解温度的差异来达到区分目的，熔解温度的差异大小即依赖于序列匹配度，同时也与碱基序列种类和排布有关。好的靶序列和合适的PCR扩增条件才能够获得好的熔解曲线区分效果。如果不然，则无法获得有差异的熔解曲线探针峰，无法达到区分的目的。
发明内容
本发明的目的在于提供一种快速鉴别PRRSV疫苗株GDr180株与其他毒株的PCR-HRM检测方法的引物及探针；
本发明的另一目的在于提供一种快速鉴别PRRSV疫苗株GDr180株与其他毒株的 PCR-HRM检测方法的试剂；
本发明的再一目的在于提供一种快速鉴别PRRSV疫苗株GDr180与其他株的PCR-HRM检测方法。
本发明所采取的技术方案是：
一种快速鉴别PRRSV疫苗株GDr180株与其他毒株的PCR-HRM检测方法的引物，其核苷酸序列如下所示：
P1:TTCTTCTTGCCTTTTCTATGCTTC；
P2:CAACTGTGTCAAGGAAATGGCT；
P3:CAGGATAGGGCATGACCGAT；
P4:CCAAATATCTCGGGATGGAACT。
一种快速鉴别PRRSV疫苗株GDr180株与其他毒株的PCR-HRM检测方法的探针，其核苷酸序列如下所示：
Probe 1:AATGTGTCAGGCACTGTGGCTGTGTG。
优选的，探针的一端利用修饰物进行修饰和封闭，修饰物包括氨基修饰的C6、反向dT、C3间臂(C3spacer)。目前来说，寡核苷酸3’端C3封闭技术比较成熟，其合成价格低廉，荧光饱和染料廉价易得而且用量少，有利于本发明技术的推广。
一种快速鉴别PRRSV疫苗株GDr180与其他株的PCR-HRM检测试剂，该试剂含有上述所述的引物和探针。
一种快速鉴别PRRSV疫苗株GDr180与其他株的PCR-HRM检测方法，包括下列步骤：
1)从待测样品中提取病毒RNA；
2)将RNA反转录成cDNA；
3)以cDNA为模板，用上述引物对P1和P2进行PCR预扩增反应，获得预扩增产物；
4)以预扩增产物作为模板，用上述引物对P3、P4以及探针Probe 1，在加入荧光饱和染料的条件下，进行不对称PCR-HRM扩增反应获得扩增产物；
5)对扩增产物进行HRM分析，确定待测样品为GDr180疫苗株或其他株。
优选的，步骤3)中PCR预扩增反应体系为：
Figure PCTCN2018088303-appb-000001
Figure PCTCN2018088303-appb-000002
优选的，步骤3)中的PCR预扩增的反应程序为：94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸35s；循环35次；72℃终延伸5min。
优选的，步骤4)中的PCR-HRM扩增反应体系为：
Figure PCTCN2018088303-appb-000003
优选的，步骤4)中的PCR-HRM扩增反应程序为：98℃变性10sec，55℃退火30sec，72℃延伸20sec，45个循环；HRM升温步骤为92℃1min，40℃2min；50℃至90℃，升温速率为0.5℃/步。
优选的，所述HRM分析方法如下所述：
1)以GDr180疫苗株不同拷贝为模板的PCR产物熔解曲线探针峰Tm值为参考，若探针峰Tm值在72.64±1.17℃，主峰Tm值在84.7±0.87℃，则判定为疫苗株GDr180；
2)若探针峰不在此范围或无探针峰的PRRSV株，则判定为PRRSV其他毒株。
本发明的有益效果是：
本发明方法结合PCR与高分辨率熔解曲线技术，筛选GDr180株的特异碱基位点，通过加入3’端封闭的寡核苷酸链，依据靶位点的序列匹配度的差异使得熔解曲线探针峰出现熔解温度的差异来达到区分目的。发明人通过反复筛选，最后找到一个GDr180株与其他疫苗株和野毒株有差异的特异基因位点，通过不对称PCR扩增来获得与探针结合的链，通过HRM分析仪获得熔解曲线，利用荧光值的导数值来分析熔解曲线的探针峰。
本发明首次建立了一种快速区分PRRSV疫苗株GDr180与其他株的PCR-HRM鉴别检测方法及引物，PCR结合HRM分析的操作简单，全部过程只需3小时，极大缩短了分型所需时间；费用低，所需探针为寡核苷酸3’端C3封闭，其合成价格低廉，荧光饱和染料廉价易得；由于实验方法特异性好，重复性好，可以准确、快速、高通量地运用于PRRSV特定疫 苗株的筛查，为快速临床诊断提供依据。
本发明的PCR-HRM引物，对GDr180与其他株均有很好地的扩增性，PCR扩增效率高，检测灵敏度高。
本发明的PCR-HRM引物特异性好，能特异性扩增PRRSV，对其他病原无扩增性，本发明对特定株的筛查具有可靠性。
附图说明
图1为PRRSV疫苗株GDr180与其他株标准样品HRM峰型化熔解曲线；
图2为PRRSV疫苗株GDr180与其他株临床样品HRM峰型化熔解曲线；
图3为PRRSV疫苗株GDr180与其他株PCR-HRM引物特异性凝胶电泳图。
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1PCR-HRM引物设计
本发明方法结合PCR与高分辨率熔解曲线技术，筛选GDr180株的特异碱基位点，通过加入3’端封闭的寡核苷酸链，依据靶位点序列匹配度的差异使得熔解曲线探针峰出现熔解温度的差异来达到区分目的。
发明人通过反复筛选，选出特异性强的引物及探针，其核苷酸序列如下所示：
P1:5'-TTCTTCTTGCCTTTTCTATGCTTC-3'(SEQ ID NO:1)；
P2:5'-CAACTGTGTCAAGGAAATGGCT-3'(SEQ ID NO:2)；
P3:5'-CAGGATAGGGCATGACCGAT-3'(SEQ ID NO:3)；
P4:5'-CCAAATATCTCGGGATGGAACT-3'(SEQ ID NO:4)；
Probe 1:5'-AATGTGTCAGGCACTGTGGCTGTGTG-C3-3'(SEQ ID NO:5)。
探针Probe 1的一端利用修饰物进行修饰和封闭，修饰物包括氨基修饰的C6、反向dT、C3间臂(C3spacer)。本发明中所用的修饰物为C3spacer，C3封闭的探针廉价且合成技术成熟，荧光饱和染料种类选择多且用量少，有利于本发明技术的推广。
其中，P1/P2引物扩增片段的长度为525bp，可以高效扩增PRRSV所有株；P3/P4引物扩增片段的长度为137bp，这一段序列包含了GDr180株与其他疫苗株和野毒株有差异的特异基因位点；Probe 1是P3/P4扩增区域的一段靶序列，通过不对称PCR扩增来获得与探针结合的链，然后进一步通过HRM分析仪获得熔解曲线，利用荧光值的导数值来分析熔解曲线的探针峰。
实施例2标准样品的制备及PCR-HRM分析
1)从样品中提取病毒RNA：
在组织匀浆器中冰上充分匀浆化处理，反复冻融3次，10,000rpm离心5min，取上清备用(有条件可以过滤除菌)。组织上清或血清采用天根Trizol试剂(天根生化)说明书进行RNA抽提。
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，本发明优先制备PRRSV GDr180株与另2株非GDr180株标准样品(包括HuN4-F112、MLV)，利用NTC作为阴性空白对照。
标准样品的制备步骤如下：分别取经过测序确定为GDr180、HuN4-F112和MLV株的质粒DNA作为模板(质粒浓度稀释至10 6copies/μL)，以P3和P4为上下游引物(质粒标准品不需要预扩增)，Probe1为封闭探针，在加有饱和荧光染料下的进行PCR扩增，其预扩增反应体系为：
Figure PCTCN2018088303-appb-000004
98℃变性10sec，55℃退火30sec，72℃延伸20sec，45个循环。HRM升温步骤为92℃1min，40℃2min；50℃至90℃，升温速率为0.5℃/步。HRM实验结果用Rotor-Gene Q TM软件进行分析。
结果见图1。3个标准品的不对称PCR扩增产物均有清晰的熔解曲线，其中GDr180株的熔解曲线探针峰Tm值为72.50℃，主熔解峰(PCR产物熔解峰)Tm值为84.60℃；非GDr180株的HuN4-F112的探针峰Tm值为64.3℃，主熔解峰Tm值为84.95℃；MLV的探针峰Tm值为65.55℃，主熔解峰Tm值为85.12℃。
因此，GDr180株的熔解曲线通过探针峰Tm的差别可以和其他标准品熔解曲线很好的区分。
对不同批次GDr180株PCR产物熔解曲线探针峰Tm与主峰Tm进行汇总，结果见表1。
表1不同批次GDr180株探针峰Tm与主峰Tm汇总
Figure PCTCN2018088303-appb-000005
根据表1的结果进行统计，GDr180疫苗株不同批次PCR产物熔解曲线探针峰Tm值为参考，探针峰Tm值在72.64±0.39℃，主峰Tm值在84.7±0.29℃。在99％置信区间内，若探针峰Tm值在72.64±1.17℃，主峰Tm值在84.7±0.87℃，则判断为疫苗株GDr180；若探针峰不在此范围或无探针峰的PRRSV株，则判断为非GDr180株的PRRSV其他毒株。
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，进行反转录成cDNA，PCR预扩增，预扩增反应体系为：
Figure PCTCN2018088303-appb-000006
94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸35s；循环35次；72℃终延伸5min。
3)以预扩增的PCR产物为模板，扩增反应体系为：
Figure PCTCN2018088303-appb-000007
98℃变性10sec，55℃退火30sec，72℃延伸20sec，45个循环。HRM升温步骤为92℃1min，40℃2min；50℃至90℃，升温速率为0.5℃/步。HRM实验结果用Rotor-Gene QTM软件进行分析。
10份样品(其中3份标准品参照，包括疫苗株GDr180、另一种疫苗株为HuN4-F112和MLV)的不对称PCR扩增产物中，7份临床样品均有清晰的熔解曲线，见图2。疫苗株GDr180标准品对照获得的熔解曲线有明显的探针峰和主峰，探针峰Tm值为72.68℃，主峰Tm值为84.8℃；HuN4-F112的探针峰Tm值为63.9℃，主峰Tm值为84.75℃，MLV的探针峰为65.65℃，主峰为85.35℃。7份临床样品中6份的探针峰Tm值为65.63±0.23℃(其中一份样品无探针峰，由于探针与靶序列差异太大)，7份样品的PCR产物熔解主峰Tm值为85.13±0.25℃。在99％置信区间，探针峰Tm值为65.63±0.69℃，与GDr180株(72.64±1.17℃)无相互交叉，探针峰Tm值差异极显著。因此7个分离株均判定为非GDr180株的PRRSV毒株。
图2的结果进行汇总，结果见表2。
表2 10份样品PCR产物熔解Tm汇总
样品名称 探针峰Tm(℃) PCR产物的熔解峰Tm(℃)
HuN4-F112 63.9 84.75
MLV 65.65 85.35
GDr180 72.68 84.8
样品1 / 84.90
样品2 65.25 84.85
样品3 65.5 85.00
样品4 65.75 85.00
样品5 65.6 85.25
样品6 65.85 85.50
样品7 65.85 85.40
由表2可知，上述分析结果和本发明的判断标准相吻合。
实施例4 PCR-HRM方法特异性实验
选择猪源性病毒PEDV、PRV、CSFV、PCV和PoRV作为阴性对照，选用P1/P2引物，结果见图3。
图3中，泳道1-5为PEDV、PRV、CSFV、PCV和PoRV，凝胶电泳显示未扩出目的条带，泳道6-7分别为PRRSV阳性参照GDr180株和HuN4-F112，有清晰目的条带，说明引物扩增的特异性好。
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 (10)

  1. 一种快速鉴别猪繁殖与呼吸综合症病毒疫苗株与其它毒株的检测方法的引物，其核苷酸序列如下所示：
    P1:TTCTTCTTGCCTTTTCTATGCTTC；
    P2:CAACTGTGTCAAGGAAATGGCT；
    P3:CAGGATAGGGCATGACCGAT；
    P4:CCAAATATCTCGGGATGGAACT。
  2. 一种快速鉴别猪繁殖与呼吸综合症病毒疫苗株与其它毒株的检测方法的探针，其核苷酸序列如下所示：
    Probe 1:AATGTGTCAGGCACTGTGGCTGTGTG。
  3. 根据权利要求2所述的探针，其特征在于：探针的一端利用修饰物进行修饰和封闭，修饰物包括氨基修饰的C6、反向dT、C3间臂。
  4. 一种快速鉴别猪繁殖与呼吸综合症病毒疫苗株与其它毒株的检测试剂，该试剂含有权利要求1所述的引物和权利要求2-3任一项所述的探针。
  5. 一种快速鉴别猪繁殖与呼吸综合症病毒疫苗株与其它毒株的检测方法，包括下列步骤：从待测样品中提取病毒RNA，将RNA反转录成cDNA；以cDNA为模板，用权利要求1所述的引物对P1和P2进行PCR预扩增反应，获得预扩增产物；
    以预扩增产物作为模板，用权利要求1所述的引物对P3、P4以及权利要求2-3任一项所述的探针Probe 1，在加入荧光饱和染料的条件下，进行不对称PCR-HRM扩增反应获得扩增产物；
    对扩增产物进行HRM分析，确定待测样品为GDr180疫苗株或其他株。
  6. 根据权利要求5所述的检测方法，其特征在于：PCR预扩增反应体系为：
    Figure PCTCN2018088303-appb-100001
  7. 根据权利要求5所述的检测方法，其特征在于：PCR预扩增的反应程序为：94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸35s；循环35次；72℃终延伸5min。
  8. 根据权利要求5所述的检测方法，其特征在于：PCR-HRM扩增反应体系为：模板 2μL
    Premix Taq HS         10μL
    引物P3       1μL
    引物P4    1μL
    探针Probe11 μL
    LC Green    1μL
    ddH 2O  余量
    总体积                20μL。
  9. 根据权利要求5所述的检测方法，其特征在于：PCR-HRM扩增反应程序为：98℃变性10sec，55℃退火30sec，72℃延伸20sec，45个循环；HRM升温步骤为92℃1min，40℃2min；50℃至90℃，升温速率为0.5℃/步。
  10. 根据权利要求5所述的检测方法，其特征在于：所述HRM分析方法如下所述：
    以GDr180疫苗株不同批次的PCR产物熔解曲线探针峰Tm值为参考，若探针峰Tm值在72.64±1.17℃，主峰Tm值在84.7±0.87℃，则判定为疫苗株GDr180；
    若探针峰不在此范围或无探针峰的PRRSV株，则判定为PRRSV其他毒株。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110438263A (zh) * 2019-08-08 2019-11-12 广东省农业科学院动物卫生研究所 一种快速鉴别prrsv基因亚型的pcr-hrm引物、检测方法与应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107099621B (zh) * 2017-05-31 2020-06-16 广东省实验动物监测所 一种快速鉴别PRRSV疫苗株GDr180株与其他毒株的PCR-HRM检测方法
CN107858455B (zh) * 2017-12-11 2021-07-20 广东省实验动物监测所 一种快速鉴别ndv疫苗株与强毒株的pcr-hrm引物及方法
CN108034765A (zh) * 2017-12-22 2018-05-15 广东省农业科学院动物卫生研究所 快速检测猪流行性腹泻病毒基因型的引物和探针、方法
CN108315483B (zh) * 2018-03-30 2021-06-25 广东省农业科学院动物卫生研究所 一种用于区分鸭坦布苏病毒野毒株和疫苗株的引物和探针的组合

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105154584A (zh) * 2015-07-14 2015-12-16 广东省农业科学院动物卫生研究所 一种快速区分prrsv经典毒株和变异毒株的hrm非标记探针方法及其引物和探针
CN105200162A (zh) * 2015-08-06 2015-12-30 广东省农业科学院动物卫生研究所 一种快速区分hp-prrs活疫苗jxa1-r株与野毒株的hrm检测方法及其引物
CN105907890A (zh) * 2016-05-05 2016-08-31 广东省农业科学院动物卫生研究所 一种快速区分HP-PRRS疫苗GDr180株与野毒株的引物、探针及方法
CN107099621A (zh) * 2017-05-31 2017-08-29 广东省实验动物监测所 一种快速鉴别PRRSV疫苗株GDr180株与其他毒株的PCR‑HRM检测方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105154584A (zh) * 2015-07-14 2015-12-16 广东省农业科学院动物卫生研究所 一种快速区分prrsv经典毒株和变异毒株的hrm非标记探针方法及其引物和探针
CN105200162A (zh) * 2015-08-06 2015-12-30 广东省农业科学院动物卫生研究所 一种快速区分hp-prrs活疫苗jxa1-r株与野毒株的hrm检测方法及其引物
CN105907890A (zh) * 2016-05-05 2016-08-31 广东省农业科学院动物卫生研究所 一种快速区分HP-PRRS疫苗GDr180株与野毒株的引物、探针及方法
CN107099621A (zh) * 2017-05-31 2017-08-29 广东省实验动物监测所 一种快速鉴别PRRSV疫苗株GDr180株与其他毒株的PCR‑HRM检测方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEE, DU ET AL.: "Genetic diversity of ORF 4-6 of type 1 porcine reproductive and respiratory syndrom virus in naturally infected pigs", VETERINARY MICROBIOLOGY, vol. 199, 21 December 2016 (2016-12-21), pages 54 - 61, XP029890755, DOI: 10.1016/j.vetmic.2016.12.026 *
ZHOU, Z. ET AL.: "Complete genomic characterization and genetic diversity of four European genotype porcine reproductive and respiratory syndrome virus isolates from China in 2011", VIRUS GENES., vol. 51, no. 3, 14 November 2015 (2015-11-14), pages 375 - 384, XP035934489, DOI: 10.1007/s11262-015-1256-z *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110438263A (zh) * 2019-08-08 2019-11-12 广东省农业科学院动物卫生研究所 一种快速鉴别prrsv基因亚型的pcr-hrm引物、检测方法与应用
CN110438263B (zh) * 2019-08-08 2023-11-17 广东省农业科学院动物卫生研究所 一种快速鉴别prrsv基因亚型的pcr-hrm引物、检测方法与应用

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