产气荚膜梭菌多重TaqMan 荧光定量PCR检测方法的建立.pdf

-0174-11网络首发时间：2 0 2 3-10-2 72024,54(02):174-184中国兽医科学2024,54(02)Chinese Veterinary ScienceD0I:10.16656/j.issn.1673-4696.2024.0023中图分类号：S852.616.3文献标志码：A文章编号：16 7 3-46 9 6(2 0 2 4)0 2产气英膜梭菌多重TaqMan荧光定量PCR检测方法的建立王景松1,2,郭亚男1*,王健霖,李继东,王建东1*（1.宁夏农林科学院动物科学研究所，宁夏银川7 50 0 0 2;2.宁夏大学动物科技学院，宁夏银川750021)摘要：为建立一种可以对产气英膜梭菌（C.perfringens)5种毒素基因cpa、c p b、e t x、i t x 和cpe进行快速检测并分型的方法，根据NCBI数据库中公布的产气英膜梭菌cpa、c p b、e t x、i t x 和cpe毒素基因序列，设计特异性引物和TaqMan探针，通过构建标准阳性质粒、优化反应条件、构建标准曲线，并进行特异性、灵敏度、重复性试验以及临床样本检测，建立可同时检测5种毒素基因的多重TaqMan荧光定量PCR方法。结果显示，建立的多重TaqMan荧光定量PCR检测方法的扩增曲线具有良好的线性关系,R0.99,90%E0.99 and 90%E 35。通过计算，各单项TaqMan荧光定量PCR的组间和组内的重复性变异系数均小于3%（表3）2.3多重TaqMan荧光定量PCR检测方法的建立2.3.1反应体系与条件的确立通过优化，确定了多重TaqMan荧光定量PCR反应体系：Taq-HSProbeqPCRPremix（U n i v e r s a l)12.5L，各引物和探针使用量见表4(引物10 mol/L,探针10 mol/L),5种重组质粒等体积混合2 L,ddH,O补至2 5L。反应参数：95预变性5min,95变性10 s,55.2退火/延伸30 s，共40 个循环，并在退火延伸后收集荧光信号。2.3.2标准曲线的建立多重TaqMan荧光定量PCR扩增曲线和标准曲线见图5。根据扩增曲线所得拷贝数(x)和C值(y)的线性关系方程式如下：pMD18-T-cpa:y=-3.308x+39.46,R=0.998 6,E=100.58%;MD18-T-cpb:y=-3.285x+37.88,R=178第54卷中国兽医科学40r3000AB2.500306200015001000y=-3.457x+39.43R=0.999 4E=94.66%1050000102030402345678模板起始拷贝数对数值循环数CycleLogarithm of startingquantity copy number40CD1 40012003076543100080020600400y=-3.457x+40.02R=0.9994E=93.99%102000C2345671020308040模板起始拷贝数对数值循环数CycleLogarithm of starting quantity copy number350040EF300030250020002015001000y=-3.448x+40.74R=0.9993E=94.99%10500C002345678010203040模板起始拷贝数对数值循环数CycleLogarithm of starting quantity copy number404000H30633000202000y=-3.466x+40.57R2=0.998 9E=94.32%101000C0OL2345678010203040模板起始拷贝数对数值循环数CycleLogarithm of starting quantity copy number900040J800070003064360005000204.0003000y=3.341x+39.97R2=0.9996E=99.21%2.000101000C002345678010203040模板起始拷贝数对数值循环数CycleLogarithm of starting quantity copy number图2 单项TaqMan荧光定量PCR扩增曲线和标准曲线Figure 2 Single-plex TaqMan qPCR amplification curve and standard curve07:标准品10 0 10 7 copies/L，下同；C：阴性对照。A、C、E、G、I:分别表示cpa、c p b、e t x、i t x 和cpe的扩增曲线；B、D、F、H、J：分别表示cpa、cpb、e t x、i t x 和cpe的标准曲线。07:Standards 10-107 copies/L,same below;C:Negative control.A,C,E,G,I:Indicate amplification curves for cpa,cpb,etx,itx and cpe,re-spectively;B,D,F,H,J:Indicate standard curves for cpa,cpb,etx,itx and cpe,respectively.179王景松等：产气荚膜梭菌多重TaqMan荧光定量PCR检测方法的建立第2 期5 000AB1550040003000100020005001 00000010203040010203040循环数Cycle循环数Cycle35004000CD30002.5003000PMHM2000200015001000100050000010203040010203040循环数Cycle循环数Cycle4000E30002.00010000010203040循环数Cycle图3单项TaqMan荧光定量PCR的特异性Figure 3 Specificity plot of single-plex TaqMan qPCRA、B、C、D、E:分别表示cpa、c p b、e t x、i t x 和cpe的特异性扩增曲线。A,B,C,D,E:Indicate the specific amplification curves for cpa,cpb,etx,itx and cpe,respectively.3500A400B300012002.5001.000200080041.500360032100024005001200CC00010203040010203040循环数Cycle循环数Cycle35004000CD300025005300052000432000150032100021000500一0C0010203040010203040循环数Cycle循环数Cycle9000E8000700060004500034000300022.000三10000C010203040循环数Cycle图4单项TaqMan荧光定量PCR的灵敏度Figure 4Single-plex TaqMan qPCR sensitivity plotA、B、C、D、E：分别表示cpa、c p b、e t x、i t x 和cpe的灵敏度扩增曲线。A,B,C,D,E:Indicate the sensitivity amplification curves for cpa,cpb,etx,itx and cpe,respectively.180第54卷中国兽医科学表3单重TaqMan荧光定量PCR的重复性试验Table 3 Single-plex TaqMan qPCR repeatability assay组间重复试验组内重复试验模板标准品浓度/（copiesL-1)Repeated testing betweengroupsRepeatedwithingroupTemplateStandard concentration平均值AVG标准差SDCV/%平均值AVG标准差SDCV/%10618.680.090.4718.670.271.47pMD18-T-cpa10523.990.612.5324.000.060.2510428.140.311.1227.860.190.7010617.840.110.6417.760.080.44pMD18-T-cpb10522.860.230.9822.830.140.6110426.120.341.3126.350.130.4810617.290.070.3917.340.090.49pMD18-T-etx10523.020.220.9722.650.010.0310426.600.140.5126.530.250.9310617.840.402.2217.720.432.40pMD18-T-itx10521.990.030.1221.900.110.5010425.720.060.2325.670.030.101018.600.110.5918.540.191.01pMD18-T-cpe10522.930.050.2222.920.130.5710426.710.070.2626.640.040.13表4多重TaqMan荧光定量PCR反应体系的引物和探针使用量Table 4Primer and probe usage of multiplex TaqManqPCR reaction systemuL靶基因引物探针Target genePrimerProbecpa0.400.40cpb0.350.35etx0.450.40itx0.300.30cpe0.350.350.998 2,E=101.56%;pMD18-T-etx:y=-3.519x+39.09,R=0.997 0,E=92.39%;pMD18-T-itx:y=3.317x+39.19,R2=0.9987,E=100.21%;pMD18-T-cpe:y=3.243x+39.97,R=0.9979,E=103.40%2.3.3特异性试验多重TaqMan荧光定量PCR特异性试验结果显示，产气荚膜梭菌A型、B型、C型、D型、E型以及各重组质粒和混合质粒均出现相对应的特异性扩增曲线,其余病原DNA和阴性对照均未有扩增曲线（图6），表明，本试验建立的产气荚膜梭菌5种毒素基因的多重TaqMan荧光定量PCR检测方法的特异性良好。2.3.4敏感性试验金多重TaqMan荧光定量PCR灵敏度试验结果显示，对等体积混合的pMD18-T-cpa、p M D 18-T-c p b、p M D 18-T-e t x、p M D 18-T-i t x 和pMD18-T-cpe标准质粒可检测到的最低拷贝数均为10 copies/L（C值 35）（图7），表明，本试验所建立的多重TaqMan荧光定量PCR灵敏度较高。2.3.5重复性试验多重TaqMan荧光定量PCR40r4000303000202000FAMy=-3.308+39.46R2=0.9986E=100.58%HEX:y=-3.285#+37.88R0.9982E=101.56%101000Cy5-5y=-3.519x+39.09R=0.9970E=92.39%Texas Redy=3.317x+39.19R=0.9987E#100.21%Cy53.243x+39.97R20.9979E103.40%0234F5678010203040模板起始拷贝数对数值循环数CycleLogarithm of starting quantity copy number图5多重TaqMan荧光定量PCR扩增曲线和标准曲线Figure55Multiple TaqMan qPCR amplification curve and standard curve主E181王景松等：产气荚膜梭菌多重TaqMan荧光定量PCR检测方法的建立第2 期400030002 00010000010203040循环数Cycle图6 多重TaqMan荧光定量PCR检测方法的特异性试验Figure 6Specificity test of multiplex TaqMan qPCR de-tection method4000300020001100010010203040循环数Cycle图7 多重TaqMan荧光定量PCR灵敏度扩增曲线Figure 7 Sensitivity amplification curve of multiplex Taq-ManqPCR衣里aqan火元量PCk里爱住试验Table5Repeatability test of multiplex TaqMan qPCR组间重复试验组内重复试验模板标准品浓度/(copiesuL-)Repeatedtesting betweengroupsRepeatedwithingroupTemplateStandardconcentration平均值ACG标准差SDCV/%平均值ACG标准差SDCV/%10620.150.1590.7920.060.1070.53pMD18-T-cpa10523.410.2010.8623.470.0550.2310427.060.1100.4127.050.0320.1210619.200.1750.9119.410.1810.94pMD18-T-cpb10522.820.2891.2722.770.0470.2110426.080.1050.4026.170.0820.3110618.060.1610.8918.180.1110.61pMD18-T-etx10521.630.2571.1921.630.0750.3510424.720.1580.6424.860.3021.2110619.560.2221.1319.720.1370.69pMD18-T-itx10523.230.1950.8423.280.0440.1910426.890.1050.3926.900.0260.1010620.720.1890.9120.870.1400.67pMD18-T-cpe10524.440.2891.1824.500.0710.3510428.130.1920.6828.150.0170.06重复性试验通过计算表明，各组内和组间的变异系数均小于2%（表5），表明，本试验所建立的多重TaqMan荧光定量PCR检测方法的重复性较好。2.3.6临床样本检测J多重TaqMan荧光定量PCR检测结果（表6)显示，在30 份菌液DNA中，30份检出cpa,1份检出cpb,18份检出etx,6份检出cpe,与产气荚膜梭菌PCR分型引物的检测结果一致。其中30 份产气荚膜梭菌阳性样品中A型11株，C型1株,D型18 株，表明，本试验所建立的方法不仅可对产气荚膜梭菌临床检测提供快速、高效的检测方法，而且可对产气荚膜梭菌进行分型和定量，具有良好的应用价值。3讨论产气荚膜梭菌是一种条件致病菌，当机体肠道环境改变时（气温、饲料、应激、疾病）可引发产气荚膜梭菌产生多种毒素，毒素穿过肠道屏障进人血液循环系统，到达各个组织器官产生损伤，导致机体发病。产气荚膜梭菌(AE)6 个毒素类型,主要引发牛羊气性坏疽、肠毒血症、肠炎等疾病,严重危害畜牧业的健康发展。通过临床样本检测可知，本研究所针对牛羊源产气荚膜梭菌A、B、C、D、E、F型的cpa、c p b、e t x、itx、c p e 毒素基因建立的五重TaqMan荧光定量PCR检测方法，通过一次反应即可对样本进行5种毒素基因检测，同时还可对样本进行定性和定量，并且一台仪器可同时检测96 个样本，1h内即可完成检测。TaqMan荧光定量PCR方法不仅弥补了多重PCR检测的耗时长、步骤繁琐等问题，同时也解决了样品检测易产生假阳性的问题。同时在样本检测过程中发现，由于产气荚膜梭菌属于肠道共生菌，临床病例粪便样本中可能存在2 种及以上产气荚182第54卷中国兽医科学表6 多重TaqMan荧光定量PCR对临床样本的检测结果Table 6 Multiplex TaqMan qPCR results for clinical samples菌液来源毒素基因Toxingene毒素分型Bacteria sourcecpacpbetxitxcpeToxin typing肉牛0 1+ABeef cattle Ol肉牛0 2+DBeef cattle 02肉牛0 3+DBeef cattle 03肉牛0 4+ABeef cattle04肉牛0 5+ABeef cattle 05肉牛0 6+DBeef cattle 06肉牛0 7+ABeef cattle 07肉牛0 8+DBeef cattle 08肉牛0 9+DBeef cattle 09肉牛10+ABeef cattle 10肉牛11+DBeef cattle ll肉牛12+ABeef cattle12肉牛13+ABeef cattle 13肉牛14+DBeef cattle 14肉牛15+ABeef cattle 15绵羊0 1+DSheep 01绵羊0 2+DSheep02绵羊0 3+CSheep 03绵羊0 4+DSheep 04绵羊0 5+DSheep 05绵羊0 6+DSheep06绵羊0 7+DSheep 07绵羊0 8+ASheep 08一绵羊0 9+DSheep09绵羊10+DSheep 10绵羊11+ASheep11一绵羊12+DSheep12绵羊13+ASheep 13绵羊14+DSheep14绵羊15+DSheep 15“十”表示阳性；“一”表示阴性。+indicates positive;indicates negative.183王景松拿夹膜梭菌多重T：荧光定量PCR检测方法的建立Van第2 期膜梭菌共同感染所致的病例,因此,在粪便DNA检测中会显示多种毒素基因检测出的结果,可利用不同毒素型的标准菌株中毒素基因的C值差作为参考，对样本进行推测是由某几种毒素型感染所致。菌液样本一般为挑取的单菌落，可以根据毒素基因的扩增曲线直接判断所属的毒素型，本研究建立的多重荧光定量PCR方法在检测毒素基因种类和灵敏度方面，cpa、c p b、e t x、i t x 和cpe质粒均可达到10 copies/L，而李一鸣等15 建立的针对产气荚膜梭菌A、B、C、D 型的cpa、c p b、e t x 毒素基因三重荧光定量PCR检测方法,其灵敏度为10 2copies/L。G u r j a r 等16 构建了产气荚膜梭菌cpa、cpb、e t x、i a、c p b 2 和cpe多毒素检测方法，其分别以cpa和cpb2、c p b 和etx以及ia和cpe这3个体系同时进行,其检测下限为57 0 pg,本试验建立的方法比其灵敏度提高10 倍。本方法不仅有良好的灵敏度、特异性和重复性，而且在同一体系中即可检测5种毒素基因,并进行6 种毒素型的分型。本试验建立的检测方法操作简单，一次加样封盖后即可，减少反复开盖带来的污染问题。同时对临床样本的检测结果实现了定性、定量及结果可视化，不仅应用前景广阔，而且为产气荚膜梭菌疾病的诊断、防控以及净化提供了技术支持。4丝结论通过对产气荚膜梭菌的5种主要毒素cpa、c p b、etx、i t x 和cpe基因分别设计5对引物和5条探针，建立可以同时检测产气荚膜梭菌5种毒素基因的多重TaqMan荧光定量PCR检测方法，其扩增曲线具有良好的线性关系；与不同病原之间无交又叉反应；检测下限为10?copies/L;变异系数均小于2%；临床样本检测具有高度可靠性，并且整个反应只需要60min即可完成。因此，本研究建立的产气荚膜梭菌多重TaqMan荧光定量PCR方法对临床样本的检测结果实现了定性、定量及结果可视化，为产气荚膜梭菌疾病的防治和净化奠定了基础。参考文献(References)1KIU R,HALL L J.An update on the human and 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