Evaluation of a new real-time PCR assay for detection of Mycobacterium scrofulaceum strains

JI Ling-yun, JIANG Yi, LIU Hai-can, WAN Kang-lin

(1. Key Laboratory of Laboratory Medicine,Ministry of Education,College of Laboratory Medicineand Life sciences, Wenzhou Medical University, Wenzhou 325035, China;2. State Key Laboratory for Infectious Diseases Prevention and Control, Collaborative Innovation Center for Diagnosisand Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, ChineseCenter for Disease Control and Prevention / National Tuberculosis Reference Laboratory, Beijing 102206, China)

Evaluation of a new real-time PCR assay for detection of Mycobacterium scrofulaceum strains

JI Ling-yun1, JIANG Yi2, LIU Hai-can2, WAN Kang-lin1

(1.KeyLaboratoryofLaboratoryMedicine,MinistryofEducation,CollegeofLaboratoryMedicineandLifesciences,WenzhouMedicalUniversity,Wenzhou325035,China;2.StateKeyLaboratoryforInfectiousDiseasesPreventionandControl,CollaborativeInnovationCenterforDiagnosisandTreatmentofInfectiousDiseases,NationalInstituteforCommunicableDiseaseControlandPrevention,ChineseCenterforDiseaseControlandPrevention/NationalTuberculosisReferenceLaboratory,Beijing102206,China)

Mycobacteriumscrofulaceum(M.scrofulaceum) is a slow-growing environmental and opportunistic atypical mycobacterium. In our study, MGB probes and specific primers were designed according toSodAgene ofM.scrofulaceum, and the sensitive, specific and rapid real-time PCR assay forM.scrofulaceumwas established, and was used to detectM.scrofulaceumin simulation samples. The minimum detectable concentration was 101copies forM.scrofulaceumDNA. The standard curve showed correlation coefficient between threshold cycle andSodAgene fragment copy number was 0.973 and slope was -3.249 which showed a good linear relationship. In addition, the minimum detectable concentration was 101Cells per milliliter for simulation sample. Also, the other nine strains showed negative results by the assay, which proved good specificity. This assay had high sensitivity and specificity for identification ofM.scrofulaceumfrom the simulation specimens. The established real-time PCR should be useful for ecological and epidemiological surveillance ofM.scrofulaceumstrains.

M.scrofulaceum; real-time PCR; nontuberculous mycobacteria

Introduction

Mycobacteria that are not members of theM.tuberculosiscomplex (M.africanum,M.bovis,M.bovisBCG,M.canettii,M.microti, andM.tuberculosis) or leprosy are classified as nontuberculous mycobacteria (NTM). NTM have been isolated from water, soil, air, food, protozoa, plants, animals, and humans[1-2]. The NTM group comprises 169 recognized species and 13 subspecies that are widely distributed in many different environments (http://www.bacterio.net/mycobacterium.html). In certain conditions (Bronchiectasis, Chronic obstructive pulmonary disease, Immunosuppressive states), NTM that are also called opportunistic mycobacteria may become pathogenic, whereas other so-called saprophytic NTM never or very rarely cause diseases[3]. However, it was reported that approximately one-third of NTM have been associated with human diseases[4-5]．

M.scrofulaceumis a slow-growing environmental and opportunistic atypical mycobacterium that causes cervical lymphadenopathy in children and disseminated infection in adult patients with immunodeficiency[6]. In addition, an extensively disseminatedM.scrofulaceuminfection was reported in an immunocompetent host with miliary lung lesions, lymphadenitis, granulomatous hepatitis, osteomyelitis, and subcutaneous abscesses[7]. In Korea,M.scrofulaceumcauses one of the most prevalent NTM pulmonary diseases, often found in immunocompetent patients[8].

A number of tools have been used in TB diagnosis, for instance smear microscopy the most widely used test has low sensitivity especially in patients with extra pulmonary tuberculosis, those with HIV co-infection and TB due to NTMs[9]. Nucleic acid amplification tests such as PCR based assay have great promise for TB diagnosis and rapid detection of drug resistance with commercial assays widely used in developed countries for over 20 years[10]. However despite their simplicity, they are prone to PCR inhibitors, some tests require post amplification procedures that increases the turn-around time and some are limited by the DNA quantities in the starting material[11-12]. Furthermore, since most of these techniques require the isolates to be cultured first, this will introduce growth competition in cases of mixed infection and hence a selection bias. Therefore accurate, rapid and cost effective methods for the identification of these NTMs and MTB are greatly needed for appropriate TB management.

Now method of Multi-locus sequence analysis (MLSA) gradually were used in identification of NTM, which usually containSodAgene. We design probe according to the Single Nucleotide Polymorphism ofM.scrofulaceumby testing the 142 kinds of NTM standard strains for 216 sequences of 541 bpSodAusing primer pairSodlgF-SodlgRdesigned after alignment of the completesodAsequences ofM.fortuitum,M.tuberculosisandM.leprae(GenBank/Entrez accession nos X70914, NC_000962 and AL450380)[13].

In our study, MGB probes and specific primers were designed according tosodAgene ofM.scrofulaceum, and the sensitive, specific and rapid real-time PCR assay forM.scrofulaceumwas established, and was used to detectM.scrofulaceumin simulation samples.

Materials and methodsDNA preparation of M．scrofulaceum strains

We used aM.scrofulaceumstrain 95017 purchased from ATCC to establish the real-time PCR method and prepare the simulation samples. The genomic DNAs of reference strains used in the experiment including nine other strains (H37Rv and eight NTM strains, see Table 1), 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). The strains were cultured using a standard Löwenstein-Jensen medium method, heat inactivated and then used directly in polymerase chain reactions (PCRs).

Tab.1 Strains used to confirm the specificity of the assay

No．StrainsH37RvM．tuberculosis95055M．bovis95016M．asiaticum95001M．avium95015M．simiae95147M．kansasii95018M．gordonae95022M．fortuitum95021M．chelonaesubsp．abscessus

Preparation of simulation samples

The inocula were prepared from actively growing bacteria collected from Lowenstein-Jensen slants or 7H10 agar plates.The inocula were adjusted with saline to a cell density of 3×108cells/mL (McFarland 1 standard). Then we diluted the inocula by the simulated sputum from 108to 100cells/mL.

Quality control

Reagents were aliquoted and each aliquot was used only once. Sterile microfuge tubes and 96 PCR well plates for real time PCR assay use. Reagent preparation, DNA extraction, DNA amplification and detection were performed in separate rooms to avoid cross contamination of amplicons.

Real-time PCR primers and probes

We designed the primers and probe by ABI primer Express 2.0 software (synthesised by), the primers were also used to prepare the standard plasmid.

Tab.2 Probe and primers for real-time PCR of M．scrofulaceum targets

Prob/PrimerSequence(5＇⁃3＇)FwdprimerCGGCGTGGTCGTCATTGRevprimerTCAACGAGATCCATCACAC⁃CAAProbeFAM⁃CGTGCCTCTTCGAG⁃MGB

Construction of standard plasmid

To determine the sensitivity of the real-time PCR method, a recombinant plasmid containing the target sequence ofM.scrofulaceumSodAgene from ATCC strain 95017 (M.scrofulaceum) was constructed as follows.

DNA was amplified with primers from 95017 strain, the fragment contains probe target sequences. The PCR products (102 bp) were cloned into the pMD-19T vector using the pMD-19T vector Cloning Kit (Takara, Dalian, China). The recombinantEscherichiacolistrain 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 serially diluted (concentration range 1.0×109-1.0×100copies/μL) to evaluate the limit of detection of the real-time PCR method.

Real-time PCR reaction conditions

Fluorescence quantitative PCR instrument was BIO RAD MJ MiniOpticon, 20 μL reaction system included: 10 μL 2×Premix Ex Taq (TaKaRa DRR390), 250 nmol/L primers, 250 nmol/L probe, 6.5 μL deionized water, and 109-100copies/μL, each 2 μL, DNA templates with different concentrations. The reaction procedures: initial denaturation 95 ℃ 10 min, then 40 cycles, 95 ℃ 15 s, 55.9 ℃ 60 s. Each operation uses three parallel samples intra-assay.

ResultsStandard curves and sensitivity

The detection range was 100-109copies/μL 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 andSodAgene fragment copy number was 0.973 and slope was -3.249 (Figure 1).

A. Sensitivity plots of primers and probes used in real-time PCR amplification ofSodAgene ofM．scrofulaceum, from 109to 100copies;B. Regression plots of primers and probes used in real-time PCR amplification ofSodAgene fragment ofM.scrofulaceum;C. Sensitivity plots of primers and probes used in real-time PCR amplification of simulation samples ofM.scrofulaceum, from 108to 100cells/mL;D. Regression plots of primers and probes used in real-time PCR amplification ofSodAgene fragment ofM.scrofulaceumin simulation samples.

Fig.1 Sensitivity and regression plots of primers and probes used in real-time PCR amplification

Repeatability

Reproducible experimental results showed that the range of intra-assay coefficient of variation was 0.90-2.05 and the range of inter-assay coefficient of variation in was 1.42-8.61 in each standard concentration point (Table 3).

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

Intra⁃assayInter⁃assayDilutionCtvalueCV(%)CtvalueCV(%)1098．187．898．011．816．927．898．188．6110815．4615．3415．891．8515．1215．5315．461．4210718．6317．8818．272．0516．5217．1918．636．1710621．821．5121．380．9920．7321．7521．802．8110525．1524．2224．132．3024．2124．7425．151．9010427．2927．0227．581．0225．7525．9827．293．1510328．4828．0228．060．9027．4827．2828．482．3110233．4632．5733．41．5031．9332．3633．462．4210137．1237．2838．311．7135．0736．0737．122．84100NNNNNNNN

Note: N, Detected negative.

Specificity

The results were positive forM.scrofulaceumin our test, and the others results were all negative for other nine strains (Table 1).

Detection of simulation samples

The detection range was 100-108cells/mL by the established assay in this study and the minimum detectable concentration was 101cells/mL. The standard curve showed correlation coefficient between threshold cycle andSodAgene fragment copy number was 0.886 and slope was -1.297. (Figure 1: C, D)

Tab.4 Amplification of SodA gene fragment of M. scrofulaceum in simulation samples

Dilution108107106105104103102101100Ctvalue22．8125．1028．7830．9532．2333．0233．2035．01N

Discussion

Immunocompetent patients with extensively disseminatedM.scrofulaceuminfection may damage the lymph nodes, lungs, liver, soft tissues, and, in all likelihood, the kidneys[7]. With increased incidence of TB and nontuberculous disease infection especially among HIV patients, diagnostics with better sensitivity and ability to identifyM.tuberculosisand nontuberculous mycobacteria are required for appropriate management.

We chose a SNP of geneSodAforM．scrofulaceumas target used in the real-time PCR. In this study, we established a rapid, sensitive and specific real-time PCR for detection ofM．scrofulaceum. The method was established by standard strain ofM.scrofulaceum, namely onlyM.scrofulaceumstandard strain had a positive result for this method，other mycobacterias were negative. And standard curve was made by plasmid with different DNA copy number (109, 108, 107, 106, 105, 104, 103, 102, 101, 100) in this experiment, we chose concentration of 105as the internal control in this experiment for the best sensitivity and specific degree. The minimum detectable concentration is 101copies forM.scrofulaceumDNA. The standard curve showed correlation coefficient between threshold cycle andSodAgene fragment copy number was 0.973 and slope is -3.249, which showed a good linear relationship. In addition, the minimum detectable concentration was 101cells/mL for simulation samples. Also, the other nine strains showed negative results by the assay, which proved good specificity.

The correlation coefficient ofM.scrofulaceumDNA is better than that of simulation samples. It is understandable as there are some inhibitors existed in simulation samples.

The limitation of this study is that we do not apply the assay to clinic samples. However, the assay is used to detect simulation samples ofM.scrofulaceumand the minimum detectable concentration reached to 101cells/mL. In conclusion, this assay had high sensitivity and specificity for identification ofM．scrofulaceumfrom the simulation specimens. The established real-time PCR should be useful for ecological and epidemiological surveillance ofM.scrofulaceumstrains.

Acknowledgments

This work was financially supported by the projects 81401647 of Natural Science Foundation of China, and 2013ZX10003006 and 2013ZX10003002-001 of Chinese National Key Program of Mega Infectious Diseases.

Authors contribution

For this study, Wan Kang-lin, Ji Ling-yun and Jiang Yi conceived and designed the experiments. Ji Ling-yun and Jiang Yi performed the experiments. Ji Ling-yun, Jiang Yi and Liu Hai-can analyzed the data. Wan Kang-lin contributed reagents, materials, and analysis tools. Ji Ling-yun and Wan Kang-lin wrote the article.

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评估一种新的检测瘰疬分枝杆菌菌株的real-time PCR方法

纪凌云1，蒋 毅2，刘海灿2，万康林1

目的 建立针对非结核分枝杆菌中瘰疬分枝杆菌菌株的real-time PCR检测方法。方法在我们的研究中,根据瘰疬分枝杆菌的SodA基因设计MGB探针和特定的引物,并用来检测模拟样品中的瘰疬分枝杆菌。结果该方法最低瘰疬分枝杆菌DNA检测浓度是101拷贝数，标准曲线显示阈值周期和SodA基因片段拷贝数之间的相关系数是0.973，斜率为-3.249,表现出良好的线性关系。此外, 模拟样品最低检测浓度是每毫升101个细菌，此外,其他9株菌为阴性结果,验证了良好的特异性。结论该方法对于检测瘰疬分枝杆菌模拟标本显示很高的敏感性和特异性，可用于瘰疬分枝杆菌菌株的生态和流行病学监测。

瘰疬分枝杆菌；real-time PCR；非结核分枝杆菌

R378.9

A

1002-2694(2016)12-1108-06

1.温州医科大学检验医学院生命科学学院,温州 325035； 2.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，感染性疾病诊治协同创新中心，北京 102206

2016-07-20；

2016-10-02

10.3969/j.issn.1002-2694.2016.012.012

Wan Kang-lin, Email: wankanglin@icdc.cn

国家重大传染病防治科技重大专项(No.2013ZX10003-006，2013ZX10003-002)和国家自然科学基金(No.81401647)联合资助