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耿震, 侯学霞, 张琳, 郝琴. 一种莱姆病螺旋体real-time PCR方法的建立及其在鼠标本检测中的应用评价[J]. 中国人兽共患病学报, 2015,31(9): 812-816
GENG Zhen, HOU Xue-xia, ZHANG Lin, HAO Qin. Evaluation of a new real-time PCR assay for detection of Borrelia burgdorferi in rodents [J]. CHINESE JOURNAL OF ZOONOSES, 2015,31(9): 812-816  
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Copyright©2015, 中国人兽共患病学报
中国人兽共患病学报
一种莱姆病螺旋体real-time PCR方法的建立及其在鼠标本检测中的应用评价
耿震, 侯学霞, 张琳, 郝琴
中国疾病预防控制中心传染病预防控制所,传染病预防控制国家重点实验室,北京 102206
郝琴,Email:haoqin@icdc.cn
收稿日期:2015-06-20
基金:十二五重大专项(No.2012ZX10004219-007 和2013ZX10004001)资助
摘要

目的 基于莱姆病螺旋体recA 基因,建立一种检测鼠中莱姆病螺旋体的real-time PCR方法。方法 通过GenBank分析比较莱姆病螺旋体recA 基因,选择其保守序列设计MGB探针及引物并进行方法学评估。并应用建立的real-time PCR方法和nested PCR方法对收集的123份鼠标本进行检测分析。结果 本研究建立的real-time PCR方法仅对莱姆病螺旋体检测阳性,其最小检出浓度为101copies/μL。标准曲线各浓度点Ct值批内、批间平均变异系数(CV)分别为1.56%和2.30%。123份鼠标本中,real-time PCR检测59例阳性,nested PCR检测43例阳性。结论 新建立的real-time PCR方法具有快速、敏感和特异的优点,可用于鼠标本中莱姆病螺旋体的检测。

关键词: 莱姆病螺旋体; real-time PCR; nested PCR; 鼠
中图分类号:R377 文献标志码:A 文章编号:1002-2694(2015)09-0812-05
Evaluation of a new real-time PCR assay for detection of Borrelia burgdorferi in rodents
GENG Zhen, HOU Xue-xia, ZHANG Lin, HAO Qin
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Correspondence author: Hao Qin, Email: haoqin@icdc.cn
Fund:Supported by the 12th Five-Year Major National Science and Technology Projects of China (Nos.2012ZX10004219-007 and 2013ZX10004001)
Abstract

We established and evaluated a real-time PCR assay for detection of Borrelia burgdorferi ( B. burgdorferi) in rodents. MGB probe and specific primers were designed based on the conserved sequences of recA gene of B. burgdorferi and methodology evaluation was performed. The 123 rodent tissue samples were detected by the established real-time PCR and nested PCR methods. Results showed real-time PCR assay based on recA gene was successfully established. Specificity evaluation showed specific amplification was only achieved from genomic DNA of B. burgdorferi. The lowest detection limit of the new assay was about 10 copies of recA gene from B. burgdorferi genomic DNA. The average intra-and-inter coefficient of variations (CV) of the Ct value were 1.56% and 2.30% respectively. In 123 rodent samples, 59 samples were detected positive by real-time PCR, compared to 43 positives by nested PCR. The new real-time PCR assay is suitable for detecting Borrelia burgdorferi in rodents.

Keywords: Borrelia burgdorferi; real-time PCR; nested PCR; rodent

Lyme disease caused by Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) is an important tick-borne infectious disease. It is very common and prevalent in temperate climate regions around the world[1, 2, 3].The clinical manifestations of Human Lyme Borreliosis (LB) include cutaneous erythema, arthritis, even serious neurological manifestations, and so on[3, 4].

In China, many studies have been done on Lyme disease since 1986, the results shows the natural foci of Lyme disease are across almost all of the forest areas in China. There are Borrelia burgdorferi infection in forest people of 29 provinces (municipalities, autonomous regions), and there are typical Lyme cases in part areas of China[5, 6, 7, 8].

Borrelia burgdorferi s.l. circulates in an enzootic cycle between reservoirs and ticks. Different species of rodents, mainly mice, have been identified to be efficient natural reservoirs for Borrelia burgdorferi s.l. However, limited study have been conducted to investigate the prevalence and distribution of Borrelia burgdorferi s.l. in rodents in China.

PCR approaches have been found to be useful for the rapid, specific and sensitive detection of B. burgdorferi, and usually was used in epidemiological investigation and surveillance of Lyme disease[4, 9, 10, 11]. Real-time PCR has features that allow for rapid detection of infectious pathogens in environmental or experimentally infected animal tissues and clinical specimens[12, 13]. This technique has been employed to detect the presence of B. burgdorferi DNA in ticks[14, 15]. But until now there is no report about this method used in testing B. burgdorferi in epidemiological investigation of rodents.

In our study, MGB probes and specific primers were designed according to recA gene of B. burgdorferi, and the sensitive, specific and rapid real-time PCR assay for B. burgdorferi was established, and was used to detect B. burgdorferi in rodent samples.

Materials and methods
DNA of reference strains

The genomic DNAs of reference strains used in the experiment including B. burgdorferi, Borrelia recurrentis, Brucella canis, Escherichia coli, Vibrio cholerae, Leptospira interrogans, Ehrlichia chaffeensis, Bartonella henselae, and Legionella pneumophila, were provided by the corresponding laboratory of National Institute for Communicable Disease Control and Prevention (ICDC), Chinese Center for Disease Control and Prevention (China CDC).

Collection of rodent samples

A total of 60 rodents were collected in Heixiazi Island in Heilongjiang Province, 32 rodents were collected in Fuliang and 31 rodents were collected in Le’ an in Jiangxi Province. The spleens and kidneys were collected in this study. DNA was extracted from spleens and kidneys by the QIAamp DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), and cryopreserved in -80 ℃ for detection.

Real-time PCR primers and probes

We designed the primers and probes by ABI primer Express 2.0 software (synthesised by Shanghai GeneCore BioTechnologies Co., Ltd.), according to the conserved sequences of B. burgdorferi provided by GenBank (B. burgdorferi-NC 001318), see Table 1. Another pair of primers were designed to prepare the standard plasmid, see Table 2.

Tab.1 Probes and primers for real-time PCR of B. burgdorferi targets
Tab.2 Primers for the preparation of standard plasmids
Construction of standard plasmid

To determine the sensitivity of the real-time method, a recombinant plasmid containing the target sequence of B. burgdorferi recA gene from Chinese standard strain PD91 (B. garinii) was constructed as follows: a 162 bp fragment was amplified with primers recA-F and recA-R from PD91 strain, the fragment contained 89 bp probe target sequences. The PCR products (162 bp) were cloned into the pMD-19T vector using the pMD-19T vector Cloning Kit (Takara, Dalian, China). The recombinant Escherichia coli strain DH-5α carrying the recombinant plasmid was inoculated on LB solid culture medium (including Ampicillin penicillin) and inoculated at 37 ℃ overnight. The recombinant plasmid DNA was extracted with a TIANpure Midi Plasmid Kit (TIANGEN, China) and validated by sequencing. The recombinant plasmid was quantified by NanoDrop Spectrophotometer ND-1000 (IMPLEN, Germany) at 260 nm and was serially diluted (concentration range 1.0 1010-1.0 100 copies/ L) to evaluate the limit of detection of the real-time method.

Real-time PCR reaction conditions

Fluorescence quantitative PCR instrument was BIO RAD MJ MiniOpticon, 20 μ L reaction system including: 10 μ L 2× Premix Ex Taq (Takara DRR390), 250 nmol/L primers, 250 nmol/L probes, 6.5 μ L deionized water, and 2 μ L DNA templates with different concentrations of 1010-100 copies/μ L. The reaction procedures: initial denaturation 94 ℃ for 2 min, then 40 cycles, 94 ℃ for 10 s, 54 ℃ for 30 s. Each operation useed three parallel samples intra-assay.

Nested PCR test

A total of 123 rodent samples were also assessed by rrf-rrl intergenic spacer nested PCR[16]. Primers of nested PCR were as follows: of the first step, the forward primer 5'CGACCTTCTTCGCCTTAAAGC3' and the reverse primer 5'TAAGCTGACTAATACTAATTACCC3'; of the second step, the forward primer 5'TCCTAGGCATTCACCATA3' and the reverse primer 5'GAGTTCGCGGGAGA3'. The PCR was performed in 50 μ L of a solution containing 8 L of extracted DNA, 1 μ mol/L of each primer and 25 μ L of 2× Taq buffer (10 mmol/L Tris-HC1, 1.5 mmol/L MgCl2, 50 mmol/L KCl, 0.01% gelatin, 200 μ mol/L of each dNTP, 1.25 U Taq MasterMix) (CWBIO, Beijing, China). A 1 L of the first PCR reaction were used as template DNA for the second PCR reaction. The PCR condition of the first step was as follows: an initial template melting step at 95 ℃ for 5 min; 35 cycles at 95 ℃ for 45 s, 53 ℃ for 45 s, and 72 ℃ for 45 s; and a final extension at 72 ℃ for 5 min. The condition the second step was the same as the first step except the annealing temperature was 55 ℃.

Results
Standard curves and sensitivity

The detection range was 101-1010copies/μ L by the established assay in this study, minimum detectable concentration was 101copies (Figure 1 and Table 3). The standard curve showed correlation coefficient between threshold cycle and recA gene copy number was 0.997 and slope was -3.271 (Figure 2).

Fig.1 Sensitivity plots of primers and probes used in real-time PCR amplification of recA gene of B. burgdorferi, from 1010 to 100copies

Fig.2 Regression plots of primers and probes used in real-time PCR amplification of recA gene of B. burgdorferi

Tab.3 Reproducibility and inter-assay coefficient of variation for the real-time PCR amplification in this study
Repeatability

Reproducible experimental results showed that the range of intra-assay coefficient of variation was 0.18%-2.78% (Table 4) and the range of inter-assay coefficient of variation was 1.09%-4.38% in each standard concentration point (Table 3).

Specificity

The results were positive for B. burgdorferi in our test, and the others results were all negative for clinical isolates, such as B. recurrentis, B. canis, E. coli, V. cholera, L. interrogans, E. chaffeensis, B. henselae, L. pneumophila, see Figure 3.

Tab.4 Reproducibility and intra-assay coefficient of variation for the real-time PCR amplification in study

Fig.3 Specific experiments of recA gene of B. burgdorferi

Detection of specimens

The 123 rodent samples had been tested by nested PCR and real-time PCR established in this study. Results showed 59 samples were tested positive by real-time PCR, and the positive rate was 47.97%; 43 rodent samples were tested positive by nested PCR, and the positive rate was 34.96%. Seventy-seven samples were tested positive by nested PCR and real-time PCR. There was statistical difference between the two methods (χ 2=4.29, 0.01< P< 0.05) (Table 5).

Results also showed the different infection rates of rodents in different locations. The positive rate of rodents in Fuliang County, Jiangxi Province was the highest (24/32, 75%), the positive rate of rodents in Heixiazi Island and Le’ an County was 58.33% and 58.06% respectively (Table 5).

Tab.5 Results of 123 rodent samples tested by nested PCR and real-time PCR
Discussion

Lyme borreliosis (LB) caused by the spirochete B. burgdorferi is a worldwide tick-borne disease. People living in the rural areas are at the highest risk of B. burgdorferi infection[17]. In China, much study on Lyme disease have been carried on since 1980’ s[4, 5, 7, 8, 9]. However, vectors and hosts are still remained unknown in some areas of China.

Several real-time methods for detection, quantification and identification of species have been reported. They based on recA, hbb, p66, or ospA. But they was mainly used to detect B. burgdorferi in ticks[14, 15, 18, 19, 20]. Until now, there is no report about real-time methods used in investigation of rodents. In this study, we established a rapid, sensitive and specific qPCR for detection of B. burgdorferi in rodents. And we also tested 123 mice samples from Jiangxi and Heilongjiang provinces to assess the established qPCR. Results showed the established real-time PCR can be used to test mice tissue samples efficiently, and it was more sensitive than nested PCR. So the established real-time PCR should be useful for ecological and epidemiological surveillance of rodents of Lyme disease.

The authors have declared that no competing interests exist.

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