Simple and rapid identification of Mycobacterium species by Real-time PCR of rpoB gene

Seong-Yong Lima, Bum-Joon Kimc, Mi-Kyung Leed, Eunhee Jeonb, Wonyong Kimb, Sang-In Chungb, and Kijeong Kimb,*aDivision of Pulmonary and Critical Care Medicine, Department of Medicine, Kangbuk Samsung Hospital, Sungkyunkwan

University School of Medicine, Seoul, South KoreabDepartment of Microbiology, College of Medicine, Chung-Ang University, Seoul 156-756, South Korea

cDepartment of Microbiology, College of Medicine, Seoul National University,, Seoul 110-799, South KoreadDeparment of Laboratory Medicine, College of Medicine, Chung-Ang University, Seoul 140-757, South Korea

IntroductionMycobacterial infectious diseases including tuberculosis require the correct species identification for the determination of an appropriate drug regimen for the treatment. Conventional methods for the identification of mycobacteria are laborious and may take several weeks. Other molecular biological methods have been developed for the rapid detection and identification of mycobacterial species but still these are inconvenient because of additionally required analysis procedures such as enzyme treatment, incubation and agarose gel electrophoresis and staining after PCR. In addition, duringsuch a long time post-PCR procedures, there is an increased risk of cross-contamination of PCR products between samples and reagents. In this presentation, we introduce a simple, rapid and non-post-PCR-procedure-requiring methods we developed to determine the species of Mycobacterium specimens by using a real-time PCR technology.

MethodsForward primer (5′-TCA AGG AGA AGC GYT ACG A) and probes for Mycobacterium species detection were designed based on the partial rpoB gene sequeunces of Genbank accession No. AM408590, BX842574, AE000516, CP000325, CP000479, AY262738, AE016958, AY147167, AY147165, AY262737, AY262736, AY147172, AY147166, CP000480, AY262735, AY147174, AY147173, AY147170, AY262739, AY262740, AY147163, AY147164). Reverse primer (5′-CGG GTT GTT CTG RTC CAT GAA -3′) and probes for NTM genus were designed based on the partial rpoB gene sequences of Genbank accession No. AF173084 - AF173088, AF057482, AF057484, AF057469, AF057470, AF057473, AF057482, AF057484, AF057454, AF057456, AF057485 - AF057496, AF057471 - AF057481, AF057483, AF057457 -AF057468, AF057455, AF057457, and AF057449 - AF05745). Oligo V 6.5 was used to design and analyze the forward and reverse primers to have similar melting temperature to each other’s, less primer duplex formation, low internal stability at the 3′ ends and high priming efficiency. To design the probes for the NTM detection at the genus level, the aforementioned rpoB gene partial sequences in which the reverse primer sequence are positioned were retrieved from the Genbank and the sequences were aligned using Multialign program developed by F. Corpet (http://bioinfo.genopole-toulouse.prd.fr/multalin/). The output result of this program was saved as the fasta format and was exactly transferred to Seqman software of DNAstar package (Fig. 1). The rpoB gene sequence of M. flavescenswas forcefully used as the consensus sequence because the sequence appeared to be one of the most homologous sequences with other species based on the alignment. Comparing others with this sequence, the most homologous region of the sequences among those of Mycobacterial species was determined. In this region that are highlighted in Fig. 1, anchor probe and sensor probe sequences were designed by using LightCycler Probe Design Software 2.0 (LC PDS 2.0) following the probe design guide presented in the software instruction manual. Briefly, probe sequences were designed trying to meet the requirements that the anchor probe and the sensor probe should be separated by no more than one to five nucleotides and anchor Tm should be 5 °C higher than the sensor Tm to ensure that the sensor probe always melts off first. In addition, avoiding inter- or intra-molecular homologies was considered. Because the anchor and sensor probe sequences for the genus detection inevitably include a couple of nucleotides which are mismatched to some target species, the anchor probes’ length was adjusted to increase the Tms of mismatched anchor probes higher than those of the paring sensor probes relying on the Tm values calculated by the software to ensure avoiding detection failure of Mycobacterium genus except M. tuberculosis. To design the Mycobacterium species-specific probes, the rpoB gene partial sequences used for the forward primer design were aligned using the Multialign program and Seqmansoftware and the sequence of a target species for its probes design was forcefully used as the consensus sequence. The species-specific regions of the target organism that appear dissimilar by more than two nucleotides at the sensor probe-binding position or four nucleotides at the anchor probe-binding position to the sequences of the respective probe binding positions of the other Mycobacterium species were searched and utilized for the probe sequence determination. A total of 20 pairs of Mycobacterium species-specific probes were designed as shown in Table 2 and Fig. 2. Probes had Tms higher than 55C which were calculated by using the LC PDS software and the Tms of the sensor probes over the matched DNA sequences of the target organisms were ensured to be sufficiently different from those over mismatched DNA sequences of the other Mycobacterium species. Three to five species detection probes were combined into five groups to use them in respective five mastermixes I to V. The probe combinations were determined to minimize inter-probe and probe-primer homologies which were analyzed by using LC PDS software. All the anchor probes were labeled by fluorescein at their 3’ end and the sensor probe for each target species in the group was labeled at its 5’ end by one of the dyes of LC Red610, LC Red640, LC Red670 and LC Red705 separately from the dyes used for the other target species probes to detect each target species via the separately sepecified detection channel. All the sensor probes were phosphorylated at their 3’ end to prevent from their modification by polymerization.

Results

Species M aster mix I probes (Calculated Tm) M aster mix II probes (C alculated Tm)

N TM M TB CHE KA N/GAS ABS GEN/SIM FO R/PER G AS

(57.5-64.4) (65.3) (72) (55.8-64.7) (68.3) (68.3) (62 .7) (62.3)

M . abscessus 58.1±0.1 - - - 67±0.1 - - -

M . avium 64.9±0.2 - - - - - - -

M . bovis - 65.8±0.2 - - - - - -

M . celatum I 61.7±0.2 - - - - - - -

M . celatum II 65.2±0.1 - - - - - - -

M . chelonae 60.9±0.3 - 69.3±0.2 - - - - -

M . flavecsens 65.7±0.2 - - - - - - -

M . fortuitum I 64.6±0.2 - - - - - 66.3±0.2 -

M . gastri 60.6±0.1 - - 59.3±0.1 - - - 66.0±0.2

M . genevanse 60.5±0.1 - - - - 69.5±0.2 - -

M . gordonae I 61±0.1 - - - - - - -

M . haem ophilum 65.7±0.3 - - - - - - -

M . intracellu lare 60.5±0.1 - - - - - - -

M . kansasii 61.9±0.2 - - 68.3±0.1 - - - -

M . m alm oense 65.8±0.1 - - - - 56.3±0.0 - -

M . m arinum 64.6±0.2 - - - - - - -

M . peregrinum 63.7±0.3 - - - - - 65.6±0.2

M . phlei 65.5±0.3 - - - - - - -

M . scrofulaceum 60.3±0.1 - - - - - - -

M . sm egmatis 65.7±0.1 - - - - - - -

M . szulgai 65.7±0.2 - - - - - - -

M . terrae 65.3±0.1 - - - - - - -

M . tuberculosis - 65.8±0.1 - - - - - -

M . ulcerans 58.2±0.2 - - - - - - -

M . xenopi 60.5±0.0 - - - -

Species Mastermix III probes (Calculated Tm) Mastermix IV probes (Calculated Tm) INT AVI PER SZU ULC/MAR TER XEN (70.4) (69.1) (670) (705) (610) (670) (705) M. abscessus - - - - - - - M. avium - 70.0±0.13 - - - - - M. bovis - - - - - - - M. celatum I - - - - - - - M. celatum II - - - - - - - M. chelonae - - - - - - - M. flavecsens - - - - - - - M. fortuitum I - - - - - - - M. gastri - - - - - - - M. genevanse - - - - - - - M. gordonae I - - - - - - - M. haemophilum - 61.7±0.13 - - - - - M. intracellulare 70.6±0.07 - - - - - - M. kansasii - - - - - - - M. malmoense - - - - - - - M. marinum - 56.2±0.07 - - 68.8±0.2 - - M. peregrinum - - 68.0±0.34 - - - - M. phlei - - - - - - - M. scrofulaceum - 56.8±0.04 - - - - - M. smegmatis 58.0±0.08 - - - 56.8±0.14 - - M. szulgai - - - 64.2±0.1 - - - M. terrae - - - - - 68.0±0.3 - M. tuberculosis - - - - - - - M. ulcerans - 56.4±0.12 - - 69.0±0.11 - - M. xenopi - - - - - - 63.4±0.21

Species Mastermix V probes (Calculated Tm) MAR GOR134 GOR2 SCR CEL1 CEL2 (60.7) (59.5, 65.5) (64.0) (63.6) (61.0) (59.8)

M. abscessus - - - - - - M. avium - - - - - - M. bovis - - - - - - M. celatum I - - - - 65.1±0.2 64.7±0.1 M. celatum II - - - - - - M. chelonae - - - - - - M. flavecsens - - - - - - M. fortuitum I - - - - - - M. gastri - - - - - - M. genevanse - - - - - - M. gordonae I - 66.2±0.2 - - - - M. haemophilum - - - - - - M. intracellulare - - - - - - M. kansasii - - - - - - M. malmoense - - - - - - M. marinum 60.3±0.0 - - - - - M. peregrinum - - - - - - M. phlei - - - - - - M. scrofulaceum - - - 67.9±0.1 - - M. smegmatis - - - - - - M. szulgai - - - - - - M. terrae - - - - - - M. tuberculosis - - - - - - M. ulcerans - - - - - - M. xenopi - - - - - -

Table 1. Measured melting temperatures (Tm , °C) of sensor probes by performing triplicate real-time PCR using LightCycler2.0 and its software for the identification of Mycobacteriumspecies

Fig. 1. Design of anchor and sensor probes for the genus identification of Nontuberculosis mycobacteria genus

Fig. 2. Design of anchor and sensor probes for the identification of Mycobacterium species. Underlines indicate the probe positions

Fig. 3. Mycobacterium identification during amplification cycles

Fig. 3. Mycobacterium identification by melting curve analysis

SummaryA real-time PCR method targeting the polymorphic regions of rpoB gene was developed for the simple, prompt and accurate identification of clinically important mycobacteria to the species level. Primers were designed to amplify about 430 bpregion of rpoB, and HybProbe formats of 6 channel Lightcycler system were adopted, and 20 pairs of oligonucleotideprobes specifically hybridizing to one Mycobacterium genus and 18 Mycobacterium species were designed. Most of these were identified by the detection of increased fluorescence signal from the specific hybridization probe biding during the amplification cycles and all of them including some non-differentiable ones were confirmatively identified by measuring their unique melting temperatures by subsequent melting-curve analysis to the species level in one round PCR. We have demonstrated that the Mycobacterium species were identified differentially, promptly and safely by this assay without any secondary labor-intensive and time-consuming analyses.

References

AcknowledgementsDohyun Myung, Seung Yun Lim and Hee Jin Kim at BMS Korea for helpful technical support.

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