A multiplex real-time PCR melting curve assay to detect drug resistant mutations of Mycobacterium tuberculosis

1 Early diagnosis of drug resistant Mycobacterium tuberculosis is urgently needed to 2 optimize treatment regimens and to prevent the transmission of resistant strains. Real3 time PCR assays have been developed to detect drug resistance rapidly, but none of them 4 have been widely applied due to their complexity, high cost, or requirement for advanced 5 instruments. In this study, we developed a real-time PCR method based on melting curve 6 analysis of dual-labeled probes. Six probes targeting rpoB 81bp core region, katG315, 7 inhA promoter, ahpC promoter and embB306 were designed and validated with clinical 8 isolates. First, 10 multi-drug resistant (MDR) strains with a wide mutation spectrum were 9 used to analyze the melting temperature (Tm) deviations of different mutations by single 10 real-time PCR. All mutations can be detected by significant Tm reductions compared to 11 the wild-type. Then three duplex real-time PCR reactions, with two probes in each, were 12 developed to detect mutations in 158 MDR isolates. Comparing with the sequencing data, 13 all mutations covered by the six probes were detected with 100% sensitivity and 100% 14 specificity. Our method provided a new way to rapidly detect drug resistant mutations of 15 M. tuberculosis. Compared to other real-time PCR methods, we use fewer probes, which 16 are labeled with the same fluorophore, guaranteeing this assay can be detected in a single 17 fluorescent channel or run on single-channel instruments. In conclusion, we have 18 developed a widely applicable real-time PCR assay to detect drug-resistant mutations of 19 M. tuberculosis. 20 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom Introduction 21 Tuberculosis (TB) is still one of the most serious threats to human health around the 22 world. Recently, the dramatic increase of drug resistant TB has caused an important 23 problem to the disease control due to the treatment collapse and the transmission of drug 24 resistant strains (1, 17). The traditional phenotypic drug susceptibility tests pose serious 25 delays in the detection of resistance due to the extremely slow growth of M. tuberculosis. 26 Rapid diagnosis of drug resistance is essential in order to initiate effective antibiotic 27 therapies and prevent the transmission of drug resistant strains. 28 M. tuberculosis acquires drug resistance mainly through mutations in specific genes (2, 29 28, 29, 33). In our previous study, a set of drug resistant mutation sites was indentified 30 for the detection of MDR TB in Shanghai, China (19). The knowledge of drug resistant 31 mutations has led to the development of molecular methods to achieve the rapid 32 diagnosis of drug resistant TB (23). Among these methods, real-time PCR has been one 33 of the most widely applied due to its rapidity, high sensitivity, reproducibility, and low 34 risk of contamination (7, 10, 12, 14, 18, 31). The most common real-time PCR assays for 35 detecting drug resistant mutations have been developed through two main methods. In the 36 first method, a fluorescent signal is generated by hybridization of a probe to the target 37 sequence at the end of each PCR cycle. The TaqMan probe and molecular beacon based 38 allele discrimination assays belong to this category. Because most of these assays require 39 the use of two different fluorophore labeled probes for one allele differentiation (9, 32), 40 the cost is relatively high and real-time PCR instrument with multiple channels is 41 required. In the second case, mutation detection is achieved by melting curve analysis. 42 Two major assays, fluorescence resonance energy transfer (FRET) probe melting curve 43 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom analysis and High Resolution Melting curve (HRM) have been successfully applied to 44 detect drug-resistant mutations of M. tuberculosis (5, 12, 16, 22, 24, 25). But, both of 45 these assays have been applied to limited real-time PCR platforms: the FRET probe assay 46 is restricted to LightCycler (Roche) and the HRM assay requires highly advanced real47 time PCR instruments. More recently, several new probe-based melting curve analysis 48 technologies, including unlabeled probes, dual-labeled probes and sloppy molecular 49 beacon have been developed for genotyping (3, 8, 34). But limited studies have reported 50 their application to detected drug resistant mutations of M. tuberculosis (4, 15). 51 In the present study, we aim to develop a low cost, widely applicable real-time PCR 52 assay, based on melting curve analysis of dual-labeled probes, to rapidly detect the drug 53 resistant mutations of M. tuberculosis. 54 55 Material and methods 56 Bacterial isolates and DNA extraction 57 The MDR M. tuberculosis isolates used for real-time PCR analysis were selected from 58 the culture collection at the Shanghai Municipal Center for Disease Control and 59 Prevention (Shanghai CDC) from March 2004 to November 2008. In total, 158 MDR 60 isolates were randomly selected from 322 MDR TB cases (19). The sequencing analysis 61 of drug-resistant genes (rpoB, katG, inhA, inhA promoter, ahpC promoter, and embB) 62 indicated the mutation pattern of these 158 strains can represent the drug-resistant 63 mutation types in Shanghai. The laboratory reference strain, M. tuberculosis H37Rv 64 which is susceptible to all drugs and which has no mutation in all drug-resistant genes, 65 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom was used as the wild-type control. DNA of M. tuberculosis isolates were extracted by a 66 rapid boiling method as previously described (26). 67 Probe and primer design 68 Six dual-labeled probes and ten primers were designed to detect mutations in five drug 69 resistance associated genes (table 1). For the 81bp rpoB core region, multiple probes 70 (normally more than four) were usually required to cover the whole region in a PCR 71 detection assay (7, 14, 31). In this study, we intended to simplify the procedure and 72 reduce the cost by designing two long probes (more than 25 nucleotides) to detect 73 mutations in this region. An internal fluorophore (FAM) was attached to the probes 74 RpoP1 and RpoP2 respectively, and quencher BHQ1 was attached at the 3’ ends of both 75 probes (table 1, figure 1A). Two mismatch nucleotides were introduced in probe rpoP2 to 76 disrupt the palindrome sequence, which could also increase the sensitivity of 77 discrimination (table 1). Other normal dual-labeled probes (fluorophore and quencher are 78 labeled at 5’ and 3’ end respectively) were designed to detect mutations in katG315, inhA 79 promoter, ahpC promoter and embB306 (table 1). All of the six probes are labeled with 80 the same fluorophore FAM. 81 Real-time PCR 82 Both single and duplex real-time PCR assays were performed in this study. The real-time 83 PCR mixture was prepared in a final volume of 20 μl. To avoid hydrolysis of probes, as 84 occurs classically during the elongation steps in TaqMan real-time PCR, aTaq DNA 85 polymerase (Promega, USA) which lacks 5’ to 3’ exonuclease activity was used for the 86 PCR reactions. The final concentration of MgCl2 was 1.5mM. The optimal concentrations 87 of primers and probes for both single and duplex PCR were listed in Table 1. To achieve 88 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom asymmetric amplification, the final concentrations of the two primers from one primer 89 pair were different with the concentration of one primer being eight folds higher than the 90 other one. One micro liter of extracted DNA was used for each reaction. For condition 91 optimization, Betaine was introduced in one group of the single real-time PCR reactions 92 with a final concentration of 1M. The real-time PCR was performed in capillary tubes in 93 Roter-Gene Q (Qiagen). The cycling conditions were denaturation at 95°C for 1 min, 94 followed by 40 cycles of amplification at 95 °C for 6 s, 58 °C for 30 s (with a single 95 acquisition of fluorescence), 72 °C for 10s, 58 °C for 15 s and 72 °C for 5s. The melting 96 program was 30 s at 95°C, 40 °C for 0 s, and 85 °C for 0 s. The rate of temperature 97 increase was 1 °C/s (or 0.5 °C/s) and fluorescence was continuously acquired. A negative 98 control without DNA sample and a standard wild-type control with DNA of H37Rv were 99 included for every real-time PCR experiment. The melting curve was analyzed using the 100 Rotor-Gen 1.7.94 software. 101 Analytical sensitivity 102 The three duplex reactions were performed on serially diluted genomic DNA (ranging 103 from 5.0×10 5 to 5.0×10 0 copies per reaction) of the wild-type strain H37Rv and a 104 MDR isolates M1 which has three mutations in rpoB531 (TCG→TTG), embB306 (ATG 105 →ATA) and katG315 (AGC→ACC). The gDNA of these two isolates was extracted by 106 the CTAB (cetyltrimethylammonium bromide) method as previously described (30). The 107 concentration of each gDNA was determined by ND-2000 UV-Vis spectrophotometer 108 (NanoDrop Technologies, Wilmington, DE). 109 110 Results 111 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom Design of the Real-time PCR assay 112 Dual-labeled probes were usually used for TaqMan real-time PCR assays. In this study, 113 six dual-labeled probes were designed to detect drug resistant mutations. In order to avoid 114 the hydrolysis of the probe by Taq polymerase in TaqMan assays, the aTaq polymerase 115 without 5’ nuclease activity was used in our assay to keep the probe intact during the 116 amplification. The fluorescent detection relies on the difference in fluorescence emission 117 between the melted and hybridized configurations of the probe (Figure 1. B). Because the 118 mean distance between the fluorophore and the quencher molecules of the melted single119 strand probe is shorter than that of the hybridized probe-amplicon duplex, a difference in 120 fluorescence emission will be readily detectable when the probes is release from its target 121 sequence (6, 8). In order to increases visibility of the probe-amplicon duplex melting 122 transition, asymmetric PCR was employed to generate excess single strand amplicons 123 which are complementary to probes. At the end of the amplification, melting curve 124 analysis was applied to detect mutations. In each run, melting curve analysis was 125 performed to both clinical isolates and wild-type control (H37Rv). Mutations were 126 detected as the Tm deviations compared to the wild-type (figure 1, C). 127 Tm deviations for mutations 128 To test the ability of our real-time PCR design to rapidly detect drug resistant mutations, 129 six independent reactions with a single probe in each were performed using 10 MDR 130 clinical strains which represented a broad spectrum of mutations based on our previous 131 sequencing data (table 2). Of the two groups of reactions, the one with 1M Betaine 132 showed a lower Tm (3 to 4 °C) for the same probe and a narrower melting peak. 133 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom Interestingly, the non-specific peak around 74°C was cleared by the addition of Betaine 134 (data not shown). So, 1M Betaine was always included in the following experiments. 135 The average reference Tms for wild-type sequences were as follows: 69.8°C for rpoP1, 136 72.9°C for rpoP2, 59.2°C for katP, 64.6°C for inhP, 67.3°C for ahpP, and 72.5°C for 137 embP (with very small standard deviations, table 2). For all 10 MDR strains, Tms of wild138 type sequences were all within the range obtained for the reference strain H37Rv. All 139 strains with mutations in rpoB 81bp core, katG315, inhA promoter, ahpC promoter and 140 embB306 were efficiently detected by the measurement of deviations in the Tms of the 141 probes compared with the value of the wild-type reference strain H37Rv. 142 For rifampin (RIF) resistance, mutations in codons 513 and 516 in rpoB were detected as 143 a result of the Tm reduction of the probe rpoP1 (both of them had a 4.4°C Tm reduction). 144 One strain with two mutations in codons 511 and 516 had a Tm reduction of 9.1°C (table 145 2). The most common mutations in codons 526 and 531 were also detected as a result of 146 the reductions in the Tms of the probe rpoP2 (Table 2). Two different types of mutations 147 in rpoB526 caused different Tm reductions (2.6 and 3.1 °C for mutation CAC→TAC and 148 CAC→CTC, respectively) and the most common mutation in rpoB codon 531(TCG→ 149 TTG) caused a Tm reduction of 3.7 °C. With regard to isoniazid (INH) resistance, all 150 mutations in katG315, inhA promoter and ahpC promoter can be efficiently detected by 151 corresponding probes. Mutations in katG315 were detected by a reduction in the Tm of the 152 probe katP (from 6.4 to 8.2°C, table 2). The mutation inhA -15 (C→T) caused a 10.5°C 153 reduction in the Tm of the probe inhP. Mutations in the ahpC promoter region were 154 detected by reductions in the Tm of probe ahpP (form 4.4 to 8.8°C), with different Tm 155 reductions indicating mutations in different locations (table 2). Mutations in embB306 156 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom were treated as a marker for MDR TB according to previous studies (13, 27). Among the 157 10 MDR strains, 5 of them with mutations in embB306 were detected by reductions in the 158 Tm of the probe embP (from 3.6 to 11.1°C) compared with the Tm of H37Rv, and the five 159 different Tm reductions indicated four different mutation types (table 2). 160 Thus, the detection of deviations in the Tms of the probes higher than 2.6°C (more than 161 ten times the standard deviation) indicated the presence of a mutation. These results were 162 reproducible by three independent experiment repeats with very small standard deviations 163 (table 2). 164 Duplex Real-time PCR and blind analysis of clinical isolates 165 In order to achieve multiplex detection of resistance mutations, three duplex real-time 166 PCR reactions, with two probes in each (table 1), were developed. The validity of this 167 duplex assay was tested with all the 158 MDR strains in a blinded manner. The reaction I, 168 which contained probe rpoP1 and rpoP2, was designed to detect mutations in rpoB 81bp 169 core region to predict RIF resistance. The melting curve analysis can clearly display the 170 Tm reductions in probe rpoP1, and although the Tm reductions in probes rpoP2 always 171 came with the overlap of the melting peaks of these two probes, the Tm reductions in 172 rpoP2 still can be detected unambiguously (figure 1a). Comparing to the sequencing data, 173 all mutations in the probe covered regions were detected with 100% sensitivity and 174 specificity. In reaction II, probe katP and inhP were used to detect mutations in katG 175 genes and inhA promoter to predict INH resistance (figure 1b). The Tm reductions in both 176 probes katP and inhP can be clearly detected by melting curve analysis, and the results 177 indicated 111 strains had mutations in katG315 and 11 strains had mutations in inhA 178 promoter region which was in 100% agreement with the sequencing data. The reaction III 179 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom which contains probe ahpP and embP was used to detect mutations in ahpC promoter 180 region and embB306 (figure 1c). In this reaction, the Tm reductions of both probes can be 181 clearly detected although melting peak overlap of the two probes happened when the 182 reduced Tm of probe embP was close to the Tm of probe ahpP. The melting curve analysis 183 successfully detected all 22 strains with mutations in ahpC promoter and 56 strains with 184 mutations in embB306. Over all, mutations within the probe covered regions were 185 detected with 100% sensitivity and 100% specificity by the three duplex real-time PCR 186 reactions. 187 Analytical sensitivity of the duplex assay 188 To assess the sensitivity of our duplex assay, we performed three reactions on serially 189 diluted DNA samples of wild-type H37Rv and MDR isolates M1. Analytical sensitivity 190 study showed that the reaction I could differentiate wild-type from mutant when the 191 concentration was as low as 5 DNA copies per reaction (figure 2, d). However, the 192 sensitivities of reaction II and III, which contain four primers respectively and were not 193 extensively optimized, were about 100 times lower than reaction I. 194 195 Discussion 196 Rapid diagnosis of drug resistance of M. tuberculosis is essential for the effective 197 treatment of patients and to prevent the dissemination of resistant strains. Among the 198 molecular methods applied to detect resistant mutations, real-time PCR has the advantage 199 of rapidity, high sensitivity, reproducibility, and low risk of contamination. Several real200 time PCR based assays including both commercial and “in house” assays have been 201 developed during the last few years (7, 9, 11, 14, 16, 18, 20, 21, 31). Most of them have 202 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom been proved high sensitivity and specificity, but due to either cumbersomeness or 203 requirement for advanced instrument, none of them have been widely applied, especially 204 in resource limited settings. 205 In the present study, we developed a low cost and widely applicable real-time PCR assay 206 to do rapid diagnosis of drug resistant M. tuberculosis. In this assay, dual-labeled probes 207 were designed to cover drug resistance related regions, and detect mutations based on the 208 melting curve analysis of the probes at the end of PCR amplification. Comparing to 209 TaqMan and molecular beacon based allele discrimination real-time PCR methods, our 210 assay has several advantages. In the TaqMan or molecular beacon based assays, two 211 probes (one pair) are usually needed to achieve the allele differentiation (9, 32). The 212 requirement to design individual probes for each resistant mutations not only increases 213 the costs but also limits these assays being only suitable for detecting those most common 214 mutations, such as those in codon 315 of katG or codon 531 of the rpoB (9). Both 215 TaqMan and molecular beacon based methods had been latterly modified to detect 216 mutations in the rpoB 81bp core region, and it has been reported that RIF-resistant M. 217 tuberculosis can be detected in a one-tube reaction by using the beacons (7, 31). But, 218 since both TaqMan probes and beacons can only cover short DNA regions, multiple 219 probes (more than four) labeled with different fluorophores would be required to fully 220 explore a region such as the rpoB 81bp core which would required a real-time PCR 221 analyzer that can detect multiple fluorescent signals simultaneously in order to achieve 222 multiple detection (7, 14, 31). In our methods, only one probe is needed to detect 223 mutations in a specific region. And for the rpoB core, two long internal fluorophore 224 labeled probes were enough to achieve the detection of mutations in this region which 225 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom reduced the cost for multiple probes. What’s more, all probes in our assay were labeled 226 with the same fluorophore (FAM) and the duplex detections were achieved by the Tm 227 difference of two probes in each reaction which guaranteed this assay can be detected in a 228 single fluorescent channel or run on single-channel instruments. 229 Similar methods based on melting curve analysis have been applied to FRET probes to 230 detect resistant mutations of M. tuberculosis (12, 16, 20). In those studies, two pairs of 231 FRET probes (sensor and anchor) were designed to detect mutations in rpoB core region. 232 But two probes were still needed to detect mutations in codon 315 of katG which caused 233 a higher cost compared to our assay. To our knowledge, FRET probe based melting curve 234 assays have been developed only for LighCycler (Roche), which is a large restriction to 235 its use on other instruments. More recently, High Resolution Melting (HRM) analysis has 236 been increasingly applied for indentifying resistant mutations (5, 22, 24, 25). HRM 237 analysis can detect small Tm shifts between wild-type and mutant samples in the melting 238 profile of the amplicons. But advanced real-time PCR instruments with high temperature 239 resolution and perfect temperature uniformity between samples are required. In our 240 methods, the Tm deviations for different mutations are bigger than 2.6°C, which can be 241 detected by almost all real-time PCR platform by melting analysis. 242 The major limitation of our assay is the overlapping of melting peaks of two probes in the 243 three duplex reactions, which increased the complexity of the melting curve analysis. 244 However, as the judgment was made by comparing the melting curve of an unknown 245 sample to the wild-type control H37Rv, the mutations can still be detected 246 unambiguously. Another limitation is the relatively low sensitivity of the duplex reaction 247 II and III which may be caused by the low efficiency of the duplex asymmetric 248 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom amplifications in these reactions. However, we believe that the sensitivity of these two 249 reactions could be improved by modifying primer sequences and concentrations. 250 In conclusion, we have developed widely applicable real-time PCR assay to detect drug 251 resistant mutation of M. tuberculosis. This new method has been proved to be efficient 252 and reliable in detecting a wide variety of mutations with clinical isolates. Further studies 253 are required to optimize the duplex assay and evaluate its performance with clinical 254 sputum specimen. 255 256 Acknowledgements 257 We are grateful to Dr. Gregory Dolganov (Stanford University School of Medicine) for 258 providing assistance in this work. 259 This work was supported by the Key Project of Chinese National Programs (No. 26