技术领域technical field
本发明涉及微生物检测技术领域，具体说是涉及一种快速、可视化并且可实时定量检测猪丁型冠状病毒的逆转录环介导等温扩增试剂盒及使用方法。The invention relates to the technical field of microbial detection, in particular to a rapid, visualized and real-time quantitative detection of porcine D-coronavirus reverse transcription loop-mediated isothermal amplification kit and its use method.
背景技术Background technique
猪丁型冠状病毒病(porcine epidemic diarrhea,PDCo)是一种由猪丁型冠状病毒(porcine epidemic diarrhea virus, PDCo V)引起的猪的一种高度接触性肠道传染病，它主要感染整段小肠，尤其是空肠和回肠，引起严重的肠炎并伴有小猪的腹泻和呕吐。Porcineepidemic diarrhea (PDCo ) is a highly contagious intestinal infectious disease of pigs caused byporcine epidemic diarrhea virus (PDCo V ), which mainly infects the entire The small intestine, especially the jejunum and ileum, caused severe enteritis with diarrhea and vomiting in piglets.
2009～2010年该病毒 PDCo V 在中国香港首次报道。猪丁型冠状病毒感染与猪流行性腹泻（PED）和猪传染性胃肠炎（TGE）的临床发病情况比较相似，可引起乳猪腹泻和呕吐，发病率和死亡率高达50%～100%，生长猪和成年猪感染后死亡率低。我国广泛存在猪流行性腹泻，2015年7月，广东某存栏2 000头基础母猪的集约化猪场5至15日龄乳猪发生PDCoV，该病传播迅速且腹泻严重，发病率 90%、死淘率 90% 以上。由于PDCo V的发病日龄小，感染率、死亡率高，因此很有必要对猪丁型冠状病毒的快速诊断方法进行研究，从而有效而快速地防控PDCo。The virus PDCoV was first reported in Hong Kong, China from 2009 to 2010. Porcine D-coronavirus infection is similar to the clinical incidence of porcine epidemic diarrhea (PED) and porcine transmissible gastroenteritis (TGE), which can cause diarrhea and vomiting in suckling pigs, and the morbidity and mortality are as high as 50% to 100%. , The mortality rate of growing pigs and adult pigs is low after infection. Porcine epidemic diarrhea is widespread in my country. In July 2015, PDCoV occurred in piglets aged 5 to 15 days in an intensive pig farm with 2,000 basic sows in Guangdong. The disease spread rapidly and had severe diarrhea. The incidence rate was 90%. The death rate is over 90%. Due to the young age of onset of PDCoV, high infection rate and high mortality rate, it is necessary to study the rapid diagnosis method of porcine D-coronavirus, so as to effectively and quickly prevent and control PDCoV.
对猪丁型冠状病毒的快速准确诊断是临床上防治猪丁型冠状病毒病的关键。目前实验室诊断PDCo V的方法为普通RT-PCR和实时荧光定量PCR的分子生物学方法，虽然PCR方法较病毒分离鉴定方法快速准确，但需要昂贵的仪器设备，成本较高，在结果判定上需要进行琼脂糖凝胶电泳，容易造成实验室污染导致出现假阳性结果，也不适合基层和现场检测。Rapid and accurate diagnosis of porcine D-coronavirus is the key to clinical prevention and treatment of porcine D-coronavirus disease. At present, the methods for laboratory diagnosis of PDCoV are ordinary RT-PCR and real-time fluorescent quantitative PCR molecular biology methods. Although the PCR method is faster and more accurate than the virus isolation and identification method, it requires expensive instruments and equipment, and the cost is high. Agarose gel electrophoresis is required, which is likely to cause laboratory contamination and lead to false positive results, and is not suitable for grassroots and on-site testing.
发明内容Contents of the invention
本发明的目的是为了解决基层检测猪丁型冠状病毒困难、耗时长和仪器昂贵等问题，提供一种为基层简便、快捷准确地检测猪丁型冠状病毒的试剂盒，为实现本发明目的所使用的技术方案为：一种检测猪丁型冠状病毒的RT-LAMP引物组，所述引物组如SEQ ID NO：1~6所示。The purpose of the present invention is to solve the problems such as difficult, time-consuming, and expensive instruments for detecting porcine D-coronavirus at the grassroots level, and to provide a simple, fast and accurate test kit for the detection of porcine D-coronavirus at the grass-roots level. The technical solution used is: an RT-LAMP primer set for detecting porcine D-coronavirus, the primer set is shown in SEQ ID NO: 1-6.
进一步地，所述引物组SEQ ID NO：1~6序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列SEQ ID NO：1~6具有相同功能的DNA分子。Further, the sequence of SEQ ID NO: 1-6 of the primer set undergoes one or several nucleotide substitutions and/or deletions and/or additions and is a DNA molecule having the same function as the sequence of SEQ ID NO: 1-6.
一种检测猪丁型冠状病毒的RT-LAMP试剂盒，所述试剂盒包括以上所述的检测猪丁型冠状病毒的RT-LAMP引物组。An RT-LAMP kit for detecting porcine D-coronavirus, said kit comprising the above-mentioned RT-LAMP primer set for detecting porcine D-coronavirus.
进一步地，所述的试剂盒还包括2×反应缓冲液、EM、超纯水和猪丁型冠状病毒RNA模板。Further, the kit also includes 2×reaction buffer, EM, ultrapure water and porcine D-coronavirus RNA template.
进一步地，所述试剂盒的反应温度为63℃，反应在15～25 分钟出现扩增。Further, the reaction temperature of the kit is 63°C, and amplification occurs within 15-25 minutes.
一种检测猪丁型冠状病毒的RT-LAMP试剂盒的应用，所述应用将试剂盒用于检测待测样本是否感染猪丁型冠状病毒，包括材料的准备、RT-LAMP引物组的设计与合成、反应模板的提取、RT-LAMP 反应体系建立以及采用RT-LAMP检测方法进行特异性检测、敏感性检测和定量检测。An application of the RT-LAMP kit for detecting porcine D-coronavirus, the application uses the kit to detect whether the sample to be tested is infected with porcine D-coronavirus, including preparation of materials, design of RT-LAMP primer set and Synthesis, extraction of reaction templates, establishment of RT-LAMP reaction system, and RT-LAMP detection method for specific detection, sensitivity detection and quantitative detection.
进一步地，所述RT-LAMP反应体系建立以25μL计，Further, the RT-LAMP reaction system was established in 25 μL,
2×反应缓冲液 12.5 μL2× Reaction Buffer 12.5 μL
EM 1 μLEM 1 μL
引物SEQ ID NO：1 5 pmolPrimer SEQ ID NO: 1 5 pmol
引物SEQ ID NO：2 5 pmolPrimer SEQ ID NO: 2 5 pmol
引物SEQ ID NO：3 40 pmolPrimer SEQ ID NO: 3 40 pmol
引物SEQ ID NO：4 40 pmolPrimer SEQ ID NO: 4 40 pmol
引物SEQ ID NO：5 20pmolPrimer SEQ ID NO: 5 20pmol
引物SEQ ID NO：6 20pmolPrimer SEQ ID NO: 6 20pmol
猪丁型冠状病毒RNA 2 μLPorcine D-coronavirus RNA 2 μL
超纯水 补足25 μL。Make up to 25 μL with ultrapure water.
进一步地，所述的RT-LAMP检测方法是采用特异性检测、敏感性检测和定量检测的方法。Further, the RT-LAMP detection method is a method of specific detection, sensitive detection and quantitative detection.
进一步地，所述的RT-LAMP检测方法采用loopamp实时浊度仪密闭进行，全程监控反应管的扩增情况。Further, the RT-LAMP detection method is carried out in a closed manner using a loopamp real-time turbidimeter, and the amplification of the reaction tube is monitored throughout the process.
本发明的实质性特点和进步是：Substantive features and progress of the present invention are:
1）特异性强1) Strong specificity
本发明的RT-LAMP检测试剂盒特异性检测的阴性对照病毒和水对照均无阳性结果出来，与PCR检测结果一致。且操作简便、快速获得检测结果、无需昂贵复杂的仪器。The negative control virus and water control of the RT-LAMP detection kit of the present invention specifically detects no positive results, which are consistent with the PCR detection results. It is easy to operate, quickly obtains test results, and does not require expensive and complicated instruments.
2）灵敏度高2) High sensitivity
普通PCR检测方法的灵敏度为2.47×10-8 ng/μL，而使用本发明的RT-LAMP检测方法，检测限约为2.47×10-10 ng/μL，是普通PCR的100倍。The sensitivity of common PCR detection method is 2.47×10-8 ng/μL, while using the RT-LAMP detection method of the present invention, the detection limit is about 2.47×10-10 ng/μL, which is 100 times that of common PCR.
3）迅速获得结果3) Get results quickly
普通的RT-PCR整个过程在24小时左右才能得出结果，目前多数建立的RT-LAMP反应方法在反应结束后，须采用琼脂糖凝胶电泳紫外线分析显像来判定结果，从病毒基因组RNA提取到获取试验结果，需要4～5小时左右。本发明提供的RT-LAMP检测方法反应在15 分钟左右出现扩增，60分钟内即可完成扩增，且结果判读方式简便，在可见光下将阳、阴性管进行比较，通过肉眼可明显看到阳性反应呈明显浑浊，阴性反应管透明；短暂离心后，阳性反应管底部有明显的白色焦磷酸镁沉淀物，而阴性反应管底部无沉淀物；或加入荧光染料，阳性反应管在紫外光下呈绿色，用肉眼便可观察实验结果。不需要再进行琼脂糖凝胶电泳紫外线分析显像来判定结果，从基因组RNA提取到获得最终结果可在2～3小时内完成。The whole process of ordinary RT-PCR can get the result in about 24 hours. At present, most established RT-LAMP reaction methods must use agarose gel electrophoresis and ultraviolet analysis and imaging to determine the result after the reaction is completed. It takes about 4 to 5 hours to obtain the test results. The reaction of the RT-LAMP detection method provided by the present invention occurs in about 15 minutes, and the amplification can be completed within 60 minutes, and the result interpretation method is simple, and the positive and negative tubes are compared under visible light, which can be clearly seen by the naked eye The positive reaction is obviously turbid, and the negative reaction tube is transparent; after a short centrifugation, there is obvious white magnesium pyrophosphate precipitate at the bottom of the positive reaction tube, while there is no precipitate at the bottom of the negative reaction tube; or add fluorescent dye, and the positive reaction tube is exposed to ultraviolet light It is green, and the experimental results can be observed with the naked eye. There is no need to perform agarose gel electrophoresis and ultraviolet analysis and imaging to determine the results, and the extraction of genomic RNA to the final results can be completed within 2 to 3 hours.
4）不造成污染4) No pollution
目前RT-LAMP方法用于直接观察的荧光染料为反应后加入，而本发明的荧光染料是在反应前加入的钙黄绿素商用染料（非syber-green），检测过程不需要开盖。此外，本发明的RT-LAMP检测方法在结果判定上，直接通过浊度仪检测反应管的浊度值来判定结果，可不进行荧光染料法检测结果或者进行琼脂糖凝胶电泳检测结果，不需要开盖，能有效避免污染。The fluorescent dye used for direct observation in the current RT-LAMP method is added after the reaction, while the fluorescent dye of the present invention is a commercial calcein dye (not syber-green) added before the reaction, and the detection process does not need to be opened. In addition, the RT-LAMP detection method of the present invention determines the result by directly detecting the turbidity value of the reaction tube through the turbidimeter, and does not need to perform the detection result of the fluorescent dye method or the detection result of the agarose gel electrophoresis, and does not need Open the cover, which can effectively avoid pollution.
5）可实时定量5) Can be quantified in real time
本发明利用Tubidimeter real-time LA-320 浊度仪来实时分析RT-LAMP反应的结果，不同的标准样品的浓度对应的浊度值的时间绘制成的标准曲线，代入标准曲线方程，即可求出各时间的猪丁型冠状病毒拷贝数，达到定量检测产物的目的。The present invention utilizes Tubidimeter real-time LA-320 turbidimeter to analyze the result of RT-LAMP reaction in real time, and the standard curve drawn by the time of the turbidity value corresponding to the concentration of different standard samples is substituted into the standard curve equation to obtain The copy number of porcine D-coronavirus at each time is obtained to achieve the purpose of quantitative detection of the product.
附图说明Description of drawings
图1是本发明的RT-LAMP方法特异性检测结果，其中1：猪丁型冠状病毒；2：猪流行性腹泻病毒；3：猪传染性胃肠炎病毒；4：猪嵴病毒；5：猪博卡病毒；6：猪细小病毒；7：猪圆环病毒2型；8：猪伪狂犬病毒；9：空白对照（水）。猪丁型冠状病毒反应管出现浊度的上升曲线，7株对照病毒反应管和水对照反应管均无扩增。Fig. 1 is the specific detection result of the RT-LAMP method of the present invention, wherein 1: porcine D-coronavirus; 2: porcine epidemic diarrhea virus; 3: porcine transmissible gastroenteritis virus; 4: porcine crest virus; 5: Porcine bocavirus; 6: porcine parvovirus; 7: porcine circovirus type 2; 8: porcine pseudorabies virus; 9: blank control (water). The rising curve of turbidity appeared in the porcine D-coronavirus reaction tube, and there was no amplification in the 7-strain control virus reaction tube and the water control reaction tube.
图2和图3是分别使用本发明RT-LAMP方法和普通RT-PCR方法进行的猪丁型冠状病毒敏感性检测的结果。其中1：2.47×101 ng/μL；2：2.47 ng/μL；3：2.47×10-1ng/μL； 4：2.47×10-2ng/μL；5：2.47×10-3ng/μL；6：2.47×10-4ng/μL；7：2.47×10-5ng/μL；8：2.47×10-6ng/μL；9：2.47×10-7ng/μL；10：2.47×10-8ng/μL；11：2.47×10-9ng/μL；12：2.47×10-10ng/μL；13：水（空白对照）。猪丁型冠状病毒基因组RNA的起始浓度为2.47×101 ng/μL，经10倍倍比连续稀释后，进行RT-LAMP和PCR扩增，结果显示本发明的RT-LAMP法检测限约为2.47×10-10 ng/μL，而普通PCR方法检测限约为2.47×10-8ng/μL。Fig. 2 and Fig. 3 are the results of the porcine D-coronavirus susceptibility detection using the RT-LAMP method of the present invention and the common RT-PCR method respectively. 1: 2.47×101 ng/μL; 2: 2.47 ng/μL; 3: 2.47×10-1 ng/μL; 4: 2.47×10-2 ng/μL; 5: 2.47×10-3 ng/μL ;6: 2.47×10-4 ng/μL; 7: 2.47×10-5 ng/μL; 8: 2.47×10-6 ng/μL; 9: 2.47×10-7 ng/μL; 10: 2.47×10-8 ng/μL; 11: 2.47×10-9 ng/μL; 12: 2.47×10 -10ng /μL; 13: water (blank control). The initial concentration of porcine D-coronavirus genomic RNA was 2.47×101 ng/μL. After 10- fold serial dilution, RT-LAMP and PCR amplification were performed. The results showed that the detection limit of the RT-LAMP method of the present invention was about It was 2.47×10-10 ng/μL, while the detection limit of common PCR method was about 2.47×10-8 ng/μL.
图4是加入荧光染料后肉眼观察结果：左管为以猪丁型冠状病毒基因组RNA为模板的反应情况，为阳性结果，右管为阴性对照的反应情况，为阴性结果。Figure 4 is the result of visual observation after adding fluorescent dye: the left tube is the reaction situation with the porcine D-coronavirus genome RNA as the template, which is a positive result, and the right tube is the reaction situation of the negative control, which is a negative result.
图5是本发明猪丁型冠状病毒定量标准曲线：利用不同的标准样品的浓度对应的浊度值对时间绘制成的标准曲线，代入标准曲线方程，即可求出各时间的猪丁型冠状病毒拷贝数。Fig. 5 is the quantitative standard curve of porcine type coronavirus of the present invention: utilize the standard curve that the turbidity value corresponding to the concentration of different standard samples draws to time, substitute into standard curve equation, can obtain the porcine type coronavirus of each time Virus copy number.
具体实施方式detailed description
1、材料的准备1. Preparation of materials
猪丁型冠状病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪嵴病毒、猪博卡病毒、猪细小病毒、猪圆环病毒2型和猪伪狂犬病毒，均为市售疫苗毒或广西兽医研究所分离鉴定和保存。RT-LAMP RNA扩增试剂盒购自北京蓝谱生物科技有限公司，货号LMP204；病毒基因组DNA/RNA提取试剂盒，购自康为世纪生物科技有限公司，货号CW0548。Porcine D-coronavirus, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine crest virus, porcine boca virus, porcine parvovirus, porcine circovirus type 2 and porcine pseudorabies virus are commercially available vaccines Poison or Guangxi Veterinary Research Institute isolated, identified and preserved. The RT-LAMP RNA amplification kit was purchased from Beijing Lanpu Biotechnology Co., Ltd., product number LMP204; the virus genome DNA/RNA extraction kit was purchased from Kangwei Century Biotechnology Co., Ltd., product number CW0548.
2 、RT-LAMP引物的设计与合成2. Design and synthesis of RT-LAMP primers
根据GenBank 中的猪丁型冠状病毒N基因序列，利用RT-LAMP法引物辅助设计软件PrimerExplorer V4 软件设计一套RT-LAMP引物，其中F3、B3 为外引物，FIP、BIP 为内引物，LF和LB为环引物，其中According to the N gene sequence of porcine tricoronavirus in GenBank, a set of RT-LAMP primers were designed using the RT-LAMP primer-assisted design software PrimerExplorer V4 software, in which F3 and B3 were outer primers, FIP and BIP were inner primers, and LF and BIP were inner primers. LB is a loop primer, where
F3 CGCAACCCCAACAATCCTF3 CGCAACCCCAACAATCCT
B3 TGCAGATTGGTCGCGTTTCB3 TGCAGATTGGTCGCGTTTC
FIP GCGTTGAAGGGGTCAACTCTGAATCAGCTGTTACCTCTCCGAFIP GCGTTGAAGGGGTCAACTCTGAATCAGCTGTTACCTCTCCGA
BIP TAGAGGAAGACCTCAGGAGCGTGCTGATTGCCTGTGCCTCBIP TAGAGGAAGACCTCAGGAGCGTGCTGATTGCCTGTGCCTC
LF GCCATCTCCGGTTGGGAALF GCCATCTCCGGTTGGGAA
LB GGAAGTGGCCCAAGATCTCA；LB GGAAGTGGCCCAAGATCTCA;
3、病毒基因组RNA提取3. Extraction of viral genome RNA
使用病毒基因组DNA/RNA提取试剂盒（康为世纪生物科技有限公司，货号CW0548）提取猪丁型冠状病毒或疑似猪丁型冠状病毒感染的肠道组织和粪便的DNA/RNA，以及对照病毒的基因组DNA/RNA。Use the virus genome DNA/RNA extraction kit (Kangwei Century Biotechnology Co., Ltd., catalog number CW0548) to extract the DNA/RNA of porcine D-coronavirus or suspected porcine D-coronavirus-infected intestinal tissue and feces, as well as the DNA/RNA of the control virus Genomic DNA/RNA.
4、RT-LAMP反应体系建立4. Establishment of RT-LAMP reaction system
按照试剂盒说明书，按25μl体系配置：According to the kit instructions, according to the 25μl system configuration:
2×反应缓冲液 12.5 μL2× Reaction Buffer 12.5 μL
EM 1 μLEM 1 μL
F3 5 pmolF3 5 pmol
B3 5 pmolB3 5 pmol
FIP 40 pmolFIP 40 pmol
BIP 40 pmolBIP 40 pmol
LF 20 pmolLF 20 pmol
LB 20 pmolLB 20 pmol
猪丁型冠状病毒RNA 5μLPorcine D-coronavirus RNA 5 μL
超纯水 补足25 μL。Make up to 25 μL with ultrapure water.
RT-LAMP反应在以实时浊度仪( LA-320C，日本荣研公司)进行密闭全程监控的形式监测本方法的检出情况，浊度仪实时监控扩增情况，可绘制标准曲线，通过获得未知样品达到0.1浊度值对应的时间值，即可从标准曲线上计算出该样品的起始拷贝数，反应温度以63℃做为反应温度。The RT-LAMP reaction monitors the detection situation of this method in the form of a real-time turbidimeter (LA-320C, Japan Eiken Co., Ltd.) for closed whole-process monitoring. The turbidimeter monitors the amplification situation in real time, and a standard curve can be drawn. By obtaining The unknown sample reaches the corresponding time value of 0.1 turbidity value, and the initial copy number of the sample can be calculated from the standard curve, and the reaction temperature is 63°C as the reaction temperature.
5、RT-LAMP检测方法5. RT-LAMP detection method
1）特异性检测1) Specific detection
使用病毒基因组DNA/RNA提取试剂盒提取猪丁型冠状病毒以及对照毒株-猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪嵴病毒、猪博卡病毒、猪细小病毒、猪圆环病毒2型和猪伪狂犬病毒的基因组DNA/RNA，作为RT-LAMP反应的模板，同时以水作为空白对照，检验RT-LAMP方法的特异性。Use the Viral Genomic DNA/RNA Extraction Kit to Extract Porcine Dicoronavirus and Control Strains - Porcine Epidemic Diarrhea Virus, Porcine Transmissible Gastroenteritis Virus, Porcine Rigivirus, Porcine Bocavirus, Porcine Parvovirus, Porcine circus Genomic DNA/RNA of virus type 2 and porcine pseudorabies virus were used as templates for the RT-LAMP reaction, and water was used as a blank control to test the specificity of the RT-LAMP method.
2）敏感性检测2) Sensitivity detection
提取的猪丁型冠状病毒基因组RNA，测定其浓度，用RNA-Free Water连续10倍倍比稀释成12个稀释度，以各RNA稀释度作为模板，进行RT-LAMP扩增和PCR扩增，对比本发明的RT-LAMP方法和普通PCR方法对检测猪丁型冠状病毒的敏感性。The extracted porcine D-coronavirus genomic RNA was measured for its concentration, and was serially diluted 10 times with RNA-Free Water to 12 dilutions, and each RNA dilution was used as a template for RT-LAMP amplification and PCR amplification. Compare the sensitivity of the RT-LAMP method of the present invention and the common PCR method for detecting porcine D-coronavirus.
3）荧光可视化检测3) Fluorescence visualization detection
根据浊度仪监控优化的条件，加入荧光染料，荧光染料在反应前加入，加入的染料为钙黄绿素商用染料，63℃下反应30分钟后，在紫外灯下观察，不采用琼脂糖凝胶电泳紫外线分析显像，避免开盖跑电泳观察造成的气溶胶污染实验室。According to the optimized conditions monitored by the turbidimeter, fluorescent dyes were added. The fluorescent dyes were added before the reaction. The dyes added were calcein commercial dyes. After reacting at 63°C for 30 minutes, observe under ultraviolet light without using agarose gel electrophoresis. Ultraviolet analysis and imaging can avoid aerosol pollution caused by opening the cover and running electrophoresis observation.
实施例1 RT-LAMP检测方法的特异性结果The specificity result of embodiment 1 RT-LAMP detection method
对1株猪丁型冠状病毒、7株对照病毒和水对照进行RT-LAMP扩增，结果如图1所示，猪丁型冠状病毒反应管在15分钟左右出现浊度的上升曲线，为阳性结果，7株对照病毒反应管和水对照反应管均无扩增情况出现，为阴性结果。RT-LAMP amplification was performed on 1 strain of porcine D-coronavirus, 7 strains of control virus and water control. The results are shown in Figure 1. The rising curve of turbidity appeared in the reaction tube of porcine D-coronavirus in about 15 minutes, which was positive. As a result, there was no amplification in the 7-strain control virus reaction tubes and water control reaction tubes, which were negative results.
实施例2 RT-LAMP检测方法的敏感性结果Example 2 Sensitivity results of RT-LAMP detection method
猪丁型冠状病毒基因组RNA的起始浓度为2.47×101 ng/μL，经10倍倍比连续稀释后，进行RT-LAMP和普通PCR扩增，结果如图2和图3所示，结果显示本发明的RT-LAMP法检测限约为2.47×10-10ng/μL，而普通PCR 法检测限为2.47×10-8ng/μL。The initial concentration of porcine D-coronavirus genomic RNA was 2.47×101 ng/μL. After 10-fold serial dilution, RT-LAMP and ordinary PCR amplification were performed. The results are shown in Figure 2 and Figure 3. The results It shows that the detection limit of the RT-LAMP method of the present invention is about 2.47×10-10 ng/μL, while the detection limit of the common PCR method is 2.47×10-8 ng/μL.
实施例3 RT-LAMP检测方法的荧光可视化检测结果Example 3 Fluorescence visualization detection results of RT-LAMP detection method
根据浊度仪监控优化的条件，加入荧光染料，63℃反应60分钟后，在紫外灯下观察，图4为观察结果，左管为以猪丁型冠状病毒RNA为模板的反应情况，为阳性结果，右管为阴性对照，为阴性结果。试验结果表明，建立的RT-LAMP方法可以方便基层使用，只需使用试剂盒配合本方法设计的RT-LAMP引物，加入样品后，用低廉的水浴锅来保持63℃ 60分钟，即可快速观察结果，且无需开盖，避免了污染。According to the optimized conditions monitored by the turbidimeter, fluorescent dyes were added, reacted at 63°C for 60 minutes, and then observed under ultraviolet light. Figure 4 shows the observation results. The left tube shows the reaction with porcine D-coronavirus RNA as a template, which is positive As a result, the right tube is a negative control, which is a negative result. The test results show that the established RT-LAMP method can be used conveniently at the grassroots level. You only need to use the kit with the RT-LAMP primer designed by this method, add the sample, and use a cheap water bath to keep it at 63°C for 60 minutes, and you can quickly observe As a result, without opening the cap, contamination is avoided.
实施例4猪丁型冠状病毒定量标准曲线的绘制The drawing of embodiment 4 porcine D-coronavirus quantitative standard curve
以猪丁型冠状病毒RNA为模板，以本RT-LAMP方法设计的外引物F3和B3为PCR扩增引物，将PCR扩增得到的目的基因片段连接于载体pMD18-T，转化大肠杆菌感受细胞DH5a，氨苄西林抗性筛选获得单克隆菌，提取重组的质粒RNA，经测序确认后，作为标准样品，测定重组质粒pMD18-T-M的起始浓度，用RNA-Free Water进行连续10倍倍比稀释12个稀释度，取各稀释度2 μL作为模板进行RT-LAMP扩增。Using porcine D-coronavirus RNA as a template, using the outer primers F3 and B3 designed by this RT-LAMP method as PCR amplification primers, the target gene fragment obtained by PCR amplification was connected to the vector pMD18-T, and transformed into E. coli competent cells DH5a, ampicillin resistance screening to obtain monoclonal bacteria, extract recombinant plasmid RNA, after sequencing and confirmation, as a standard sample, determine the initial concentration of recombinant plasmid pMD18-T-M, and perform serial 10-fold dilution with RNA-Free Water For 12 dilutions, 2 μL of each dilution was used as a template for RT-LAMP amplification.
设置对照：浓度为2.474×101 ng/μL、2.474 ng/μL、2.474×10-1 ng/μL、2.474×10-2 ng/μL、2.474×10-3ng/μL、2.474×10-4 ng/μL、2.474×10-5 ng/μL、2.474×10-6ng/μL、2.474×10-7ng/μL、2.474×10-8ng/μL和2.474×10-9 ng/μL的标准重组质粒pMD18-T-M样品各一个，因为浓度的负对数值与浊度值为0.1的时间值成线性关系，所以可以将浊度仪捕捉到的值与时间（如表1）做成标准曲线，获得标准曲线方程，y=0.4611x-8.059，如图5所示。从标准曲线方程来看相关系数R2为0.9959，呈良好的线性关系。以时间为X值，可求出Y值即浓度的负次方数，则浓度为10-y，再乘以基数2.474，即为2.474 ×10-y ng/μL。依据拷贝数换算公式copies/μL=（6.02 × 1023× (ng/ul × 10-9)） / (RNA length ×660)，RNAlength为载体序列大小加上目的基因序列大小，为2693+191=2884 bp，将其换算成拷贝数（copies/μL）：6.02 × 1023 ×（2.474 ×10-y×10-9）/ (2884 × 660)，简化为：7.82 ×108×10-y。如某试验样品达到浊度值为0.1的时间为25分钟时，带入所建立的标准曲线方程，求出Y等于3.4685，则浓度为10-3.4685，再乘以基数2.474，即为该试验样品的浓度2.474×10-3.4685ng/μL，拷贝数为7.82 ×108 ×10-3.4685，即为7.82 ×104.5315 copies/μL，从而达到定量的效果。Set the control: the concentration is 2.474×101 ng/μL, 2.474 ng/μL, 2.474×10-1 ng/μL, 2.474×10-2 ng/μL, 2.474×10-3 ng/μL, 2.474×10-4 ng/μL, 2.474×10-5 ng/μL, 2.474×10-6 ng/μL, 2.474×10-7 ng/μL, 2.474×10-8 ng/μL and 2.474×10-9 ng/μL standards One sample of recombinant plasmid pMD18-TM, because the negative logarithmic value of the concentration is linearly related to the time value when the turbidity value is 0.1, so the value captured by the turbidimeter and time (as shown in Table 1) can be made into a standard curve, Obtain the standard curve equation, y=0.4611x-8.059, as shown in Figure 5. According to the standard curve equation, the correlation coefficient R2 is 0.9959, showing a good linear relationship. Taking time as the X value, the Y value can be obtained, that is, the negative power of the concentration, then the concentration is 10-y , and then multiplied by the base 2.474, which is 2.474 × 10-y ng/μL. According to the copy number conversion formula copies/μL=(6.02 × 1023 × (ng/ul × 10-9 )) / (RNA length ×660), RNAlength is the vector sequence size plus the target gene sequence size, which is 2693+191= 2884 bp, converted to copy number (copies/μL): 6.02 × 1023 × (2.474 × 10-y × 10-9 )/ (2884 × 660), simplified to: 7.82 × 108 × 10-y . If it takes 25 minutes for a certain test sample to reach the turbidity value of 0.1, bring it into the established standard curve equation, and find that Y is equal to 3.4685, then the concentration is 10-3.4685 , and then multiplied by the base number 2.474, which is the test sample The concentration is 2.474×10-3.4685 ng/μL, and the copy number is 7.82 ×108 ×10-3.4685 , which is 7.82×104.5315 copies/μL, so as to achieve quantitative effect.
表1 样品浓度负对数值与PDCO V-LAMP浊度值时间线性关系表Table 1 The time linear relationship between the negative logarithm value of sample concentration and the turbidity value of PDCO V-LAMP
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等，均应包含在本发明的包含范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention shall be included within the scope of the present invention.
序列表sequence listing
<110> 广西壮族自治区兽医研究所<110> Veterinary Research Institute of Guangxi Zhuang Autonomous Region
<120>一种检测猪丁型冠状病毒的RT-LAMP引物组、试剂盒及应用<120>A RT-LAMP primer set, kit and application for detecting porcine D-coronavirus
<130>2017<130>2017
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<170>PatentIn version 3.3<170>PatentIn version 3.3
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<213> 人工序列（Artificial sequence Latin）<213> Artificial sequence Latin
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<213> 人工序列（Artificial sequence Latin）<213> Artificial sequence Latin
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<223>对人工序列的描述：外引物B3<223> Description of the artificial sequence: outer primer B3
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<212> DNA<212>DNA
<213> 人工序列（Artificial sequence Latin）<213> Artificial sequence Latin
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<223>对人工序列的描述：内引物FIP<223> Description of artificial sequences: internal primer FIP
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<213> 人工序列（Artificial sequence Latin）<213> Artificial sequence Latin
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710761229.0ACN107460255A (en) | 2017-08-30 | 2017-08-30 | A kind of RT LAMP primers group, kit and application for detecting pig fourth type coronavirus |
| Application Number | Priority Date | Filing Date | Title |
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| CN201710761229.0ACN107460255A (en) | 2017-08-30 | 2017-08-30 | A kind of RT LAMP primers group, kit and application for detecting pig fourth type coronavirus |
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| CN107460255Atrue CN107460255A (en) | 2017-12-12 |
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| CN201710761229.0APendingCN107460255A (en) | 2017-08-30 | 2017-08-30 | A kind of RT LAMP primers group, kit and application for detecting pig fourth type coronavirus |
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