改良单叠氮丙啶-荧光定量PCR法的建立及其检测抗结核药物活性的价值

doi:10.3969/j.issn.1000-6621.2020.05.011

摘要/Abstract

摘要：

目的建立改良单叠氮丙啶(propidium monoazide xx, PMAxx)-荧光定量PCR(qPCR)鉴定MTB活菌和热灭活菌的方法,并探讨PMAxx-qPCR进行抗结核药物体外活性检测的价值。方法通过活菌和热灭活菌优化 PMAxx针对MTB菌株H37Rv的预处理条件,包括曝光时间、暗孵育时间、PMAxx浓度,以及靶标DNA片段长度等。通过绘制量效曲线和建立循环阈值(Cq)与菌负荷的标准曲线,应用PMAxx-qPCR法计算测定异烟肼(INH)、利福平(RFP)的体外抗菌活性,评价检测敏感度,并分别与微孔板Alamar Blue(MABA)法和菌落形成单位(CFU)计数法进行比较,菌落计数采用独立样本t检验进行统计分析,以P<0.05为差异有统计学意义。结果当细菌负荷设定为10 ⁷CFU/ml时,在PMAxx浓度为10μmol/L、暗孵育10min后曝光15min可有效鉴别活菌、死菌(ΔCq_死-活=6.772±0.453),且使用>200bp靶标片段可更有效地反映活菌、死菌的差异。PMAxx-qPCR方法在药物作用后3d可获得与MABA法一致性良好的最低抑菌浓度(MIC)检测结果(INH:0.049~0.076μg/ml与0.032~0.064μg/ml,t=0.782,P=0.491;RFP:0.102~0.145μg/ml与0.051~0.079μg/ml,t=2.828,P=0.066)。定量标准曲线Cq与菌计数(lgCFU/ml)呈良好的线性关系(R ²=0.9863),最低检测限为10 ²CFU/ml,且PMAxx-qPCR方法和CFU计数方法测算16×MIC、8×MIC、4×MIC、2×MIC和1×MIC浓度的INH的抗菌计数分别为(4.376±0.344)和(4.325±0.318)、(4.232±0.106)和(3.936±0.194)、(4.122±0.277)和(3.874±0.105)、(3.950±0.113)和(3.675±0.250)、(3.770±0.228)和(3.618±0.257)CFU/ml,两种方法计数比较差异均无统计学意义(t值分别为0.165、1.894、1.186、1.419和0.626,P值分别为0.880、0.199、0.357、0.292和0.595),而16×MIC、8×MIC、4×MIC、2×MIC和1×MIC浓度的RFP的抗菌计数分别为(4.577±0.216)和(4.675±0.250)、(4.445±0.054)和(4.374±0.675)、(3.627±0.173)和(3.154±0.076)、(1.946±0.359)和(2.159±0.083)、(1.552±0.423)和(0.960±0.202)CFU/ml,两种方法计数比较差异均无统计学意义(t值分别为0.469、0.199、3.535、0.784、1.777,P值分别为0.671、0.855、 0.071、0.490、0.174)。结论建立的PMAxx-qPCR方法稳定,可应用于一线抗结核药物INH和RFP的活性检测,敏感度高且快速准确。

关键词: 单叠氮丙啶, 抗结核药, 聚合酶链反应, 微生物敏感性试验, 对比研究

Abstract:

Objective To establish the method of modified propidium monoazide (PMAxx)-quantitative PCR (qPCR) for identifying live and heat-inactivated Mycobacterium tuberculosis (MTB), and evaluate the value of PMAxx-qPCR assay to detect activity of antituberculosis drugs in vitro. Methods The pre-treatment conditions of PMAxx including light-exposure time, dark-incubation time, PMAxx concentration and DNA amplification size were optimized with live and heat-inactivated MTB (H37Rv). Through plotting the dose-effect curve and establishing the standard curve of the quantification cycle (Cq) value and bacterial load, the antibacterial activity of isoniazid (INH) and rifampin (RFP) in vitro were calculated by this assay, the detection sensitivity was evaluated, and comparing it to Microplate Alamar Blue assay (MABA) and colony forming unit (CFU) method, the colony count was analyzed by independent sample t-test, and the difference was statistically significant (P<0.05). Results When the bacterial load was set as 10 ⁷ CFU/ml, the dead and living bacteria could be identified effectively (ΔCq_dead-live=6.772±0.453) under the condition of the concentration of PMAxx with 10 μmol/L, dark incubation for 10 min and light-exposure for 15 min. The difference of the dead and living bacteria could be confirmed more effectively by using the >200 bp target DNA fragments. PMAxx-qPCR method obtained good MIC results comparing to MABA method in three days (INH: 0.049-0.076 μg/ml vs. 0.032-0.064 μg/ml, t=0.782, P=0.491; RFP: 0.102-0.145 μg/ml vs. 0.051-0.079 μg/ml, t=2.828,P=0.066). The Cq value of the quantitative standard curve showed a good linear relationship with the CFU (lgCFU/ml) (R ²=0.9863), and the limit number of detection was 10 ²CFU/ml. Meanwhile, the antibacterial counts of 16×, 8×, 4×, 2× and 1×MIC concentrations of INH by the PMAxx-qPCR method and CFU method were (4.376±0.344) and (4.325±0.318) CFU/ml, (4.232±0.106) and (3.936±0.194) CFU/ml, (4.122±0.277) and (3.874±0.105) CFU/ml, (3.950±0.113) and (3.675±0.250) CFU/ml, (3.770±0.228) and (3.618±0.257) CFU/ml, respectively. The antibacterial counts of 16×, 8×, 4×, 2× and 1×MIC concentrations of RFP by the PMAxx-qPCR method and CFU method were (4.577±0.216) and (4.675±0.250) CFU/ml, (4.445±0.054) and (4.374±0.675) CFU/ml, (3.627±0.173) and (3.154±0.076) CFU/ml, (1.946±0.359) and (2.159±0.083) CFU/ml, (1.552±0.423) and (0.960±0.202) CFU/ml respectively, all differences were not statistically significant ((t=0.165, P=0.880; t=1.894, P=0.199; t=1.186, P=0.357; t=1.419, P=0.292; t=0.626, P=0.595) and (t=0.469,P=0.671; t=0.199,P=0.855; t=3.535,P=0.071; t=0.784,P=0.490; t=1.777,P=0.174)). Conclusion The established PMAxx-qPCR method in this study is stable. It can be used to detect the activity of the first-line antituberculous drugs including INH and RFP with high sensitivity, rapid and accuracy.

Key words: Propidium monoazide (PMA), Antitubercular agents, Polymerase chain reaction, Microbial sensitivity tests, Comparative study

张静, 陈曦, 王彬, 付雷, 陆宇, 陈效友. 改良单叠氮丙啶-荧光定量PCR法的建立及其检测抗结核药物活性的价值[J]. 中国防痨杂志, 2020, 42(5): 472-480. doi: 10.3969/j.issn.1000-6621.2020.05.011

ZHANG Jing, CHEN Xi, WANG Bin, FU Lei, LU Yu, CHEN Xiao-you. Establishment of modified propidium monoazide (PMAxx)-quantitative PCR assay and its application for identification of antituberculosis drug activity[J]. Chinese Journal of Antituberculosis, 2020, 42(5): 472-480. doi: 10.3969/j.issn.1000-6621.2020.05.011

图/表 10

表1

表2

曝光时间对PMAxx-qPCR方法鉴定活菌和热灭活菌的影响

曝光时间	活菌Cq值( $x ˉ$ ±s)	q值	P值	热灭活菌Cq值( $x ˉ$ ±s)	q值	P值
对照组	26.910±0.458	3.199^a	0.198	27.130±0.402	11.860^a	0.000
(未经PMAxx处理)		2.357^b	0.475		18.830^b	0.000
		2.515^c	0.412		16.810^c	0.000
		8.975^d	0.000		19.110^d	0.000
曝光10min	27.850±0.149	0.842^e	0.974	30.615±1.085	6.963^e	0.001
		0.684^f	0.988		4.950^f	0.017
		5.776^g	0.005		7.241^g	0.001
曝光15min	27.603±0.736	0.158^h	0.999	32.661±0.066	2.013^h	0.620
		6.618ⁱ	0.001		0.278ⁱ	0.999
曝光20min	27.649±0.182	6.460^j	0.002	32.070±0.466	2.291^j	0.502
曝光30min	29.547±0.422	-	-	32.743±0.208	-	-

表2

表3

暗孵育时间对PMAxx-qPCR方法鉴定活菌和热灭活菌的影响

暗孵育时间	活菌Cq值( $x ˉ$ ±s)	q值	P值	热灭活菌Cq值( $x ˉ$ ±s)	q值	P值
对照组	26.910±0.458	2.635^a	0.103	27.130±0.402	13.760^a	0.000
(未经PMAxx处理)		2.127^b	0.262		12.540^b	0.000
		3.991^c	0.006		12.110^c	0.000
暗孵育10min	27.971±0.275	0.508^d	0.997	32.670±0.135	1.222^d	0.806
		1.356^e	0.726		1.649^e	0.531
暗孵育30min	27.767±0.244	1.864^f	0.397	32.178±0.291	0.427^f	0.999
暗孵育60min	28.517±0.078	-	-	32.006±0.598	-	-

表3

图1

图2

表4

PMAxx处理前后qPCR所测循环阈值(Cq值)在不同生长周期结核分枝杆菌中的比较

检测方法	生长周期2周	生长周期3周	生长周期4周
qPCR(Cq值, $x ˉ$ ±s)	26.844±0.376	27.173±0.127	27.509±0.177
PMAxx-qPCR(Cq值, $x ˉ$ ±s)	27.419±0.523	28.503±0.428	30.375±0.127
t值	1.546	5.160	22.810
P值	0.197	0.007	0.000

表4

图3

图4

图5

表5

不同MIC倍增浓度的INH、RFP作用10d后的PMAxx-qPCR法抗菌计数和CFU计数结果的比较

药物浓度	INH				RFP
药物浓度	CFU计数法 (CFU/ml, $x ˉ$ ±s)	PMAxx-qPCR法 (CFU/ml, $x ˉ$ ±s)	t值	P值	CFU计数法 (CFU/ml, $x ˉ$ ±s)	PMAxx-qPCR法 (CFU/ml, $x ˉ$ ±s)	t值	P值
16×MIC	4.325±0.318	4.376±0.344	0.165	0.880	4.675±0.250	4.577±0.216	0.469	0.671
8×MIC	3.936±0.194	4.232±0.106	1.894	0.199	4.374±0.675	4.445±0.054	0.199	0.855
4×MIC	3.874±0.105	4.122±0.277	1.186	0.357	3.154±0.076	3.627±0.173	3.535	0.071
2×MIC	3.675±0.250	3.950±0.113	1.419	0.292	2.159±0.083	1.946±0.359	0.784	0.490
1×MIC	3.618±0.257	3.770±0.228	0.626	0.595	0.960±0.202	1.552±0.423	1.777	0.174

表5

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引物名称	序列	基因位点	目的片段长度(bp)
rrs1	F5'-GGTCAACTCGGAGGAAGGTG-3'	1155~1174	81
	R5'-CGGCCATTGTAGCATGTGTG-3'	1216~1235
rrs2	F5'-TCGTGTCGTGAGATGTTGGG-3'	1059~1078	117
	R5'-CCACCTTCCTCCGAGTTGAC-3'	1156~1175
rrs3	F5'-AATTCCTGGTGTAGCGGTGG-3'	664~683	143
	R5'-GTTTACGGCGTGGACTACCA-3'	787~806
rrs4	F5'-ACGGAAAGGTCTCTTCG-3'	66~82	206
	R5'-CTTGGTAGGCCGTCAC-3'	256~271
rrs5	F5'-ACTGAGATACGGCCCAGACT-3'	318~337	279
	R5'-TCACGAACAACGCGACAAAC-3'	577~596
rrs6	F5'-AATTCCTGGTGTAGCGGTGG-3'	664~683	316
	R5'-CCAGGTAAGGTTCTTCGCGT-3'	960~979
rrs7	F5'-TACTGCAGGGGAGACTGGAA-3'	646~665	433
	R5'-CCCAACATCTCACGACACGA-3'	1059~1078