US20140162260A1 - Primers, snp markers and method for genotyping mycobacterium tuberculosis - Google Patents

Primers, snp markers and method for genotyping mycobacterium tuberculosis Download PDF

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US20140162260A1
US20140162260A1 US14/089,990 US201314089990A US2014162260A1 US 20140162260 A1 US20140162260 A1 US 20140162260A1 US 201314089990 A US201314089990 A US 201314089990A US 2014162260 A1 US2014162260 A1 US 2014162260A1
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Shih-Feng Tsai
Chien-Hsing Lin
Horng-Yunn DOU
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National Health Research Institutes
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

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  • the present invention relates to the primers, the SNP markers and the method for detecting Mycobacterium tuberculosis , and more particularly for genotyping M. tuberculosis.
  • Tuberculosis is a worldwide healthcare concern. It has been characterized by the World Health Organization (WHO) as an epidemic and estimated that one-third of the world's population has been infected with Mycobacterium tuberculosis (MTB). Epidemiologic studies have revealed that various genotypes of M. tuberculosis (MTB) may be prevalent in different geographic regions and that genotype distribution is associated with population migrations. Whether MTB genomic diversity influences human disease in clinical settings remains an open question.
  • WHO World Health Organization
  • MTB Mycobacterium tuberculosis
  • H37Rv strain MTB The complete genome of H37Rv strain MTB was published in 1988, which has a length of about 4 Mb and contains about 4000 genes. MTB can be classified as six major strains and 15 subordinate strains. Genomic variations affect the transmission, virulence, antimicrobial resistance and other attributes of the MTB, so that the development of molecular techniques for differentiating various MTB isolates is of considerable interest in epidemiological studies.
  • Genotyping methods aiming at generating phylogenetically informative data have been developed to investigate multiple clinical samples from different sources.
  • Spoligotyping is based on polymorphisms in the direct repeat (DR) locus, which is consisted of 36-bp DR copies interspaced by non-repetitive spacer sequence. It is a PCR-based reverse hybridization technique for MTB genotyping.
  • DR direct repeat
  • VNTRs variable number tandem repeats
  • MIRUs mycobacterial interspersed repetitive units
  • the present application describes a primer set for genotyping M. tuberculosis selected from one of the group consisting of primer sets 1-25.
  • the present application provides an extension primer for genotyping M. tuberculosis selected from one of the group consisting of SEQ ID Nos. 51-75.
  • the present application provides a combination of single-nucleotide polymorphism markers of M. tuberculosis selected from the group consisting of “T” at position 301 of SEQ ID No.76, “A” at position 301 of SEQ ID No. 77, “A” at position 301 of SEQ ID No. 78, “G” at position 301 of SEQ ID No. 79, “G” at position 301 of SEQ ID No. 80, “G” at position 301 of SEQ ID No. 81, “C” at position 301 of SEQ ID No. 82, “G” at position 301 of SEQ ID No. 83, “C” at position 301 of SEQ ID No. 84, “A” at position 301 of SEQ ID No.
  • the present application also provides a method for genotyping M. tuberculosis comprising obtaining a sample, amplifying and obtain at least one of first DNA fragment by using one or more primer sets selected from the group consisting of primer sets 1 to 25 (SEQ ID Nos. 1 to 50), amplifying and obtain at least one of second DNA fragment by using the obtained first DNA fragment as template and using one or more extension primers selected from the group consisting of SEQ ID Nos. 51 to 75, and detecting the second DNA fragment by using mass spectrometry.
  • the present application also provides a kit for genotyping M. tuberculosis comprising at least one primer set selected from the group consisting of primer sets 1 to 25 (SEQ ID Nos. 1 to 50), and at least one extension primer selected from the group consisting of SEQ ID Nos. 51 to 75.
  • FIG. 1 is an overall scheme for selecting lineage-specific DNA markers.
  • FIG. 2 illustrates linkage disequilibrium of SNP markers in the MTB genomes.
  • the LD plot was created using Haploview software, and the color code on plot followed the standard color scheme for Haploview: blue indicates
  • 1 and LOD ⁇ 2, and bright red indicates
  • 1 and LOD ⁇ 2.
  • FIG. 3 illustrates phylogenetic analysis of MTB isolates using strain-specific SNP markers.
  • Phylip software was applied to calculate the Nei's distance using 110-SNP (A) and 25-tagSNP (B) data, and then constructed phylogenetic trees using the neighbor joining approach, and
  • FIG. 3 also illustrates typed SNP position on MTB chromosome: 110-SNP (C) and 25-tagSNP (D).
  • FIG. 4 shows identification of specific markers for strain typing.
  • FIG. 5 illustrates decision tree based on four lineage-specific SNP markers.
  • Four of 32 lineage-specific SNPs with 100% variant allele frequencies were used to classify 81 clinical isolates into ancient Beijing (Ba), modern Beijing (Bm), East African-Indian (EAI) and Latin American and Mediterranean (LAM) lineage.
  • FIG. 6 shows high genetic diversity within Euro American lineage.
  • A Phylogenetic analysis of Euro American strains using 4,419 whole-genome SNP markers. Phylogenetic tree was constructed based on Nei's distance using the Phylip software (neighbor joining approach).
  • B Principal component analysis (PCA) of Euro American strains. The genotype data of 4,419 whole-genome SNPs was transformed into numeric values, and then PCA method was applied to analyze these 14 clinical Euro American isolates and H37Rv reference strain using SAS program.
  • PCA Principal component analysis
  • FIG. 7 shows a minimum spanning tree based on 24-MIRU-VNTR genotyping of 156 Mycobacterium tuberculosis isolates.
  • the circles represent different types classified by 24-MIRU-VNTR genotypes and were colored according to the spoligotype classification.
  • the sizes of circles represent the number of isolates with a particular genotype. ( ⁇ : indicate misclassified by spoligotyping).
  • FIG. 8 shows a new hypothetic subtype definition of Euro American lineage.
  • Phylogenetic tree was constructed based on Nei's distance using the Phylip software (neighbor joining approach).
  • FIG. 9 shows high genetic homozygosity within new hypothetic Euro American subtypes.
  • Fourteen Euro American strains (7 Haarlem and 7 T strains) were genome-wide sequenced using 454 or HiSe2000 sequencer, and there were two major clusters (6 and 7 belong to EuAm1 and EuAm2 subtypes, respectively) of them based on phylogenetic tree ( FIG. 8 ).
  • There were 81 EuAm1-specific and 133 EuAm2-specific SNPs with variant allele frequency 100%.
  • FIG. 10 shows the PCR primers, the extension primers, the positions and the correspondent alleles for the 25 tagSNPs with the multiplex reaction well scheme.
  • FIG. 11 shows the comparisons of the present application and the conventional genotyping methods.
  • the primer set for genotyping M. tuberculosis is selected from the group consisting of primer sets 1-25, each primer set contains a forward primer and a reverse primer.
  • the primer sets 1-25 are shown as follows:
  • Primer set 1 (SEQ ID No. 1) ACGTTGGATGTTCTGGACGACCTGTCCTAC and (SEQ ID No. 2) ACGTTGGATGAGCTGCGCCAAGGTTCGTG, Primer set 2: (SEQ ID No. 3) ACGTTGGATGTTGTAGCTGCCCAAATTGCC and (SEQ ID No. 4) ACGTTGGATGGGCTTCAATCTCGGCTTGG, Primer set 3: (SEQ ID No. 5) ACGTTGGATGTATTCAACACCGGCATCGGG and (SEQ ID No. 6) ACGTTGGATGTCGCCTGGTCGTGGAAGAAC, Primer set 4: (SEQ ID No. 7) ACGTTGGATGATCGGACAGCAGAAGGCAC and (SEQ ID No.
  • Primer set 12 (SEQ ID No. 23) ACGTTGGATGGTTGTTTTTGGCCGGGCAG and (SEQ ID No. 24) ACGTTGGATGATCGAGCAGACTCAGCGCTT, Primer set 13: (SEQ ID No. 25) ACGTTGGATGTGCTACCGCCAATGTTCAAC and (SEQ ID No. 26) ACGTTGGATGATGGCGTTGACATAACTCGG, Primer set 14: (SEQ ID No. 27) ACGTTGGATGATAGCAAGCACGATTGCGAC and (SEQ ID No. 28) ACGTTGGATGACCCCCCGCTGAGGGCGTA, Primer set 15: (SEQ ID No.
  • Primer set 19 (SEQ ID No. 37) ACGTTGGATGACAACCGGCCGCAGCGTTT and (SEQ ID No. 38) ACGTTGGATGAAGAACACCGAAAGTGGCTG, Primer set 20: (SEQ ID No. 39) ACGTTGGATGTGCATTGGCCACTAAAGCTC and (SEQ ID No. 40) ACGTTGGATGTCGATGACTATCTGCGGATG, Primer set 21: (SEQ ID No. 41) ACGTTGGATGACCCATTTGCCGAACGTGTC and (SEQ ID No. 42) ACGTTGGATGTGCTTGGCGACTTTGTGCAG, Primer set 22: (SEQ ID No.
  • the primer set can be applied in polymerase chain reaction to amplify a DNA fragment containing a single-nucleotide polymorphism (SNP) of M. tuberculosis .
  • SNP single-nucleotide polymorphism
  • the above primer sets can be used alone or in combination.
  • the combination of the primer sets can be applied simultaneously in one test tube for PCR test.
  • any combination selected from the primer sets 1-12 can be applied simultaneously.
  • any combination selected from the primer sets 13-22 can be applied simultaneously.
  • any combination selected from the primer sets 23-25 can be applied simultaneously.
  • extension primer for genotyping M. tuberculosis is selected from SEQ ID Nos. 51-75.
  • the extension primers of the present application are listed as follows:
  • the extension primer can be applied in polymerase chain reaction to amplify a DNA fragment having a single-nucleotide polymorphism (SNP) of M. tuberculosis as a terminal nucleotide of the DNA fragment.
  • SNP single-nucleotide polymorphism
  • the above primers can be used alone or in combination. In some embodiments, the combination of the above primer can applied simultaneously in one test tube for PCR test.
  • the SNP markers of M. tuberculosis are selected from: “T” at position 301 of SEQ ID No. 76, “A” at position 301 of SEQ ID No. 77, “A” at position 301 of SEQ ID No. 78, “G” at position 301 of SEQ ID No. 79, “G” at position 301 of SEQ ID No. 80, “G” at position 301 of SEQ ID No. 81, “C” at position 301 of SEQ ID No. 82, “G” at position 301 of SEQ ID No. 83, “C” at position 301 of SEQ ID No. 84, “A” at position 301 of SEQ ID No.
  • SNP markers of M. tuberculosis are correspondent to “T” at position 128290 of genome of the reference strain, “A” at position 178812 of genome of the reference strain, “A” at position 243118 of genome of the reference strain, “G” at position 374353 of genome of the reference strain, “G” at position 375095 of genome of the reference strain, “G” at position 430332 of genome of the reference strain, “C” at position 756840 of genome of the reference strain, “G” at position 848652 of genome of the reference strain, “C” at position 991896 of genome of the reference strain, “A” at position 996219 of genome of the reference strain, “A” at position 1300047 of genome of the reference strain, “A” at position 1810066 of genome of the reference strain, “G” at position 1932201 of genome of the reference strain, “A” at position 2008738 of genome of the reference strain, “G” at position 2165256 of genome of the reference strain, “G” at position 2165554 of genome of
  • the reference strain is M. tuberculosis H37Rv having a complete genome sequence NC — 000962.2.
  • NCBI National Center for Biotechnology Information
  • Table 1 shows the detail information of the SNP markers of the present application.
  • the H37Rv genome position indicates the position of each SNP marker in the reference chromosome
  • the reference allele indicates the nucleotide exist in M. tuberculosis H37Rv strain
  • the variant allele is the SNP marker of the present application.
  • various genotypes of M. tuberculosis possess various combinations of the SNP markers.
  • the combination of the SNP markers contains at least two markers, such as 3, 5, 7, 10, 15, 20, or 25 markers.
  • the above SNP markers may used alone.
  • the present application also provides a method for genotyping M. tuberculosis comprising obtaining a sample, amplifying and obtain at least one of first DNA fragment by using one or more primer sets selected from the group consisting of primer sets 1 to 25 (SEQ ID Nos. 1 to 50), amplifying and obtain at least one of second DNA fragment by using the obtained first DNA fragment as template and using one or more extension primers selected from the group consisting of SEQ ID Nos. 51 to 75, and detecting the second DNA fragment by using mass spectrometry.
  • the method can further comprises analyzing the mass spectrometry data based on the single-nucleotide polymorphism markers selected from Table 1.
  • the mass spectrometry is matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
  • Examples of the suitable sample applied for the method can include, without limitation, bacterial culture, nasal mucus, phlegm saliva, blood, section of tissues or organ, biopsy and the like.
  • the present application further provides a kit for genotyping M. tuberculosis comprising at least one primer set selected from the group consisting of primer sets 1 to 25 (SEQ ID Nos. 1 to 50), and at least one extension primer selected from the group consisting of SEQ ID Nos. 51 to 75.
  • the kit may further comprises a database of genotypes of M. tuberculosis based on single-nucleotide polymorphism markers, preferably, based on the SNP markers shown in Table 1.
  • the inventors sequenced the genome of six strains isolated in Taiwanese population using next-generation DNA sequencers (Roche 454/Illumina GAIIx). Based on the comparative genome analysis, there were 60 and 141 strain-specific single nucleotide polymorphisms (SNPs) found in PE/PPE and non-PE/PPE gene families, respectively, comparing to the H37Rv reference strain. In the present application, lineage specific SNPs were used as markers to design a novel strain classification scheme and conduct the phylogenetic analyses. The performance of this genotyping panel was compared with the current standard test, spoligotyping patterns specific for 156 Mycobacterium tuberculosis complex (MTBC) isolates.
  • MTBC Mycobacterium tuberculosis complex
  • MTB isolates were collected between 2004 and 2007 from the mycobacteriology laboratories of five general hospitals located in four geographical regions in Taiwan, namely, Taipei Tri-Service General Hospital (northern region), Mennonite Christian Hospital (eastern region), Wan-Ciao Veterans Hospital (central region), Tainan Chest Hospital (southern region), and Kaohsiung Veterans General Hospital (southern region).
  • the bacterial strains used in this study are representative of the diversity of MTB in Taiwan as shown previously (Chang J R et al., Clin Microbiol Infect 2011, 17(9):1391-1396; Dou H Y et al., BMC Infect Dis 2008, 8:170; Dou H Y et al., Infect Genet Evol 2008, 8(3):323-330; Dou H Y et al., J Microbiol Methods 2009, 77(1):127-129). Spoligotyping and MIRU-VNTR genotyping assays were performed based on internationally standardized protocols. A total of 156 isolates (of the Beijing, EAI, Haarlem, LAM, T, MANU, and unclassified strains) that had all genotype data available were used for the subsequent analyses.
  • TB strains were sequenced 14 to 28-fold depth of the genome separately using a Genome Sequencer 20 (GS-20) or a Genome Sequencer FLX (GS-FLX) instrument (454 Life Sciences, Roche) with a 500-800 base-pair shotgun library for each strain.
  • GS-20 Genome Sequencer 20
  • GS-FLX Genome Sequencer FLX
  • DNA libraries of six MTB Haarlem and six T clinical isolates were prepared using Nextera DNA sample preparation kit (Illumina, CA, USA), and were multiplex sequenced (2 ⁇ 100 bp) at one lane of flow cell using HiSeq2000 sequencer. After performing de-multiplex procedure, the average sequence size of each sample was 3.38 Gb, and the depths of these samples were ranged from 568 to 1068-fold when mapping to H37Rv reference sequence, resulting in that the reference coverage of these samples was from 99.44% to 99.82%.
  • the 454 sequencing raw data (sff files) from each strain were collected into a specific folder as the read source to align the reference genome of the strain H37Rv. H37Rv genome sequence and the annotated gene information were downloaded from the NCBI ftp site for Microbial Genome Assembly/Annotation Projects (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/ Mycobacterium — tuberculosis _H37Rv_uid57777/).
  • 454 GS Reference Mapper (Roche) software version 2.3) was used to map 454 reads to the reference sequence (see Table 2 for detail information) and generate high-confidence variations between the reference and each of our six MTB clinical strains.
  • Strain-specific (observed only in single strain) SNPs were selected and grouped into two categories: PE/PPE protein family and non-PE/PPE. According to the location of the variations, they can be synonymous or non-synonymous to the coding sequences. And in non-PE/PPE group, the variations can also locate at non-coding sequences, which are intergenic regions. To further confirmation using MassARRAY Analyzer (Sequenom), the number of the variations was reduced with criteria that both total depth and variation depth must larger than 15 and the variation frequency must larger than 90% for each variation site.
  • PCR and extension primers were designed for 60 PE/PPE and 60 randomly-selected non-PE/PPE SNPs using the MassArray Assay Design 3.1 software (Sequenom, San Diego, Calif.). Five of them were excluded due to difficult sequences. PCRs contained, in a volume of 5 ul per well, 1 pmol of the corresponding primers, 5 ng genomic DNA, and HotStar reaction Mix (Qiagen) in 384-well plates. Three wells were needed for each sample. PCR conditions were as follows: 94° C. for 15 min, followed by 40 cycles of 94° C. (20 s), 56° C. (30 s), 72° C. (60 s), and a final extension of 72° C. for 3 min.
  • each sample was denatured at 94° C., followed by 40 cycles of 94° C. (5 s), 52° C. (5 s), 72° C. (5 s).
  • the mass spectrum from time-resolved spectra was retrieved by using a MassARRAY mass spectrometer (Sequenom), and each spectrum was then analyzed using the SpectroTYPER software (Sequenom) to perform the genotype calling.
  • the clustering patterns of five SNPs could not be used to correctly perform genotype calling, and the data of 110 SNPs (57 PE/PPE and 53 non-PE/PPE) were finally used in the following analyses.
  • the Lewontin D′ measure was used to estimate the intermarker coefficient of linkage disequilibrium (LD) as shown in FIG. 2 .
  • An extra stringent criteria, r2 1 between each pair markers, was used to select 25 tagSNPs from 110 SNPs.
  • A27, A18, M24 were sequenced by the 454 FLX sequencer with longer average read length of 227 base-pair and fewer runs of sequencing experiments.
  • the sequencing depths were about 14X ⁇ 23X in 454 GS20 data and about 16X ⁇ 28X in 454 FLX data.
  • mapping results were summarized in Table 3. All six isolates got at least 95.8% of mapped reads that covered 97% and above of the reference sequence.
  • the total contig numbers for the three isolates sequenced by 454 GS20 were 214-305; while for the three isolates sequenced by 454 FLX they were 290-299.
  • the base quality of Phred score 40 and above (Q40Bases) for large contigs were 99.48% to 99.95% in the six isolates, indicating that the sequencing quality is high enough.
  • HC high-confidence
  • MNPs multiple nucleotide polymorphisms
  • INDELs insertions and deletions
  • SNPs co-exist in all the six isolates, and 13, 19, 232, 538 SNPs exist in five, four, three, and two of the six isolates, respectively (details were shown in Table 4).
  • the most abundant SNPs are 2,376 strain-specific (HC differences exist only in one of the six strains) and we used them as candidates for seeking lineage-specific SNPs.
  • These candidate SNPs according to their locations in coding or non-coding regions, are divided into three main categories: PE/PPE gene family, non-PE/PPE gene family, and intergenic SNPs (as shown in Table 2).
  • non-synonymous SNPs seem to have more or equal number them synonymous SNPs, except in M7 isolate.
  • PE/PPE comprises about 10% of the coding capacity of the TB genome, thus the SNPs in PE/PPE family are commonly much less than those in other non-PE/PPE gene families.
  • A18 belongs to the EAI lineage, has 4-10 times higher numbers of specific SNPs than other five isolates, suspects that the lineage may evolve at a higher mutation rate and quickly adapt to changes in their host environment.
  • 120 lineage-specific SNPs were selected to design primers for Sequenom MassArray assays. These 120 lineage-specific SNPs were unequally selected from six lineage samples as shown in Table 5, which was caused by the difference in the total numbers of lineage-specific SNP between them. These 120 SNPs were divided into two categories: [5] all of 60 SNPs within PE/PPE gene family; [8] 60 of 1,215 non-synonymous SNPs within in non-PE/PPE gene family (details were shown in Table 6).
  • phylogenetic trees were constructed based on 110-SNP or 25-tagSNP information as shown in FIG. 3(A) and FIG. 3(B) .
  • the positions of 110-SNP and 25-tagSNP were shown in FIG. 3(C) and FIG. 3(D) .
  • the morphology of 25-tagSNP phylogenetic tree was the same as that of 110-SNP tree, indicating that 25 tagSNPs can well represent the genomic variances between strains.
  • EAI and 3 LAM strains were grouped into the corresponding branches in both phylogenetic trees.
  • each of these 32 SNPs can be used to represent MTB lineage of ancient Beijing, modern Beijing, EAI and LAM, decision tree was constructed based on four lineage-specific SNP markers ( FIG. 5 ). Based on the decision tree, 75 of 107 (70%) spoligotype-classified isolates can be correctly grouped into the corresponding lineage, and 6 of 49 (10.1%) spoligotype-unclassified isolates were grouped into known lineage.
  • the 25 tagSNPs can well represent the genomic variants between strains.
  • PCR and extension primers for 25 tagSNPs were designed using the MassArray Assay Design 3.1 software (Sequenom, San Diego, Calif.). The PCR primers, the extension primers, the positions and the correspondent alleles for the 25 tagSNPs are shown in FIG. 10 .
  • PCRs contained, in a volume of 5 ul per well, 1 pmol of the corresponding primers, 5 ng genomic DNA, and HotStar reaction Mix (Qiagen) in 384-well plates. Three wells were needed for each sample. PCR conditions were as follows: 94° C. for 15 min, followed by 40 cycles of 94° C. (20s), 56° C. (30s), 72° C.
  • each sample was denatured at 94° C., followed by 40 cycles of 94° C. (5s), 52° C. (5s), 72° C. (5s).
  • the mass spectrum from time-resolved spectra was retrieved by using a MassARRAY mass spectrometer (Sequenom), and each spectrum was then analyzed using the Sequenom Typer 4.0 software (Sequenom) to perform the SNP genotype calling.
  • Tuberculosis remains a major public health issue in Taiwan and throughout the world. Over the past years, the development of genotyping methods for molecular epidemiology study of tuberculosis has advanced our understanding of the transmission of MTB in human populations. Classification of strains into sub-lineages provides perspective on the phenotypic consequences of genetic variations of the MTB strains. Phylogenic analyses of MTB strains have also offered new insights regarding the evolution of MTB and the existence of distinct clades. From public health perspective, an ideal methodology to determine the genetic variation of MTB clinical isolates should be simple, affordable, have a rapid turnaround time, and the result should be transferable in a format that can be easily shared between laboratories.
  • Strain-specific SNP typing can provide precise sequence-based information, and could be automated for large-scale studies of molecular epidemiology and phylogenetics.
  • the combination of spoligotyping and MIRU-typing can be considered a cost-effective method for TB genotyping.
  • the spoligotype is still only about 20-40% strains that cannot be sorted, and nothing in this law to compensate for this shortcoming.
  • the proposed MIRU-VNTR typing method could not sufficiently differentiate M. tuberculosis strains comprising many Beijing genotype strains. Therefore, this typing method could not be used for routine epidemiological study in areas where the Beijing genotype is prevalent.
  • the addition of several VNTR loci is required to use VNTR typing as a routine epidemiological tool without doing RFLP analysis.
  • M. tuberculosis isolates are essential for understanding the dynamics of transmission. Genetic information will help determine precise quantitative measures for transmission dynamics and augment classical epidemiological models. The ability to assess the inter-strain genetic relationships provides a powerful means of resolving a number of epidemiological issues, such as tracing of chains of transmission, determining sources of infection, differentiating recent transmission from reactivation and reinfection from relapse or treatment failure, detecting laboratory cross-contaminations, monitoring the geographic distribution and spread of particular genetic strains (including those of special epidemiological importance), or investigating the evolution of M. tuberculosis.
  • epidemiological issues such as tracing of chains of transmission, determining sources of infection, differentiating recent transmission from reactivation and reinfection from relapse or treatment failure, detecting laboratory cross-contaminations, monitoring the geographic distribution and spread of particular genetic strains (including those of special epidemiological importance), or investigating the evolution of M. tuberculosis.
  • the proposed workflow of selecting lineage-specific DNA marker ( FIG. 1 ) is an effective and logistical way to discriminate MTB isolates into genetic subtypes. Importantly, the concept of our workflow is also applicable in other fields of microbial projects, e.g., searching highly conserved domains of variable clinical isolates for vaccine development.
  • FIG. 11 shows the comparisons of the present application and the conventional genotyping methods.
  • the sample needed for the detection is as low as 20 ng of DNA sample for PCR.
  • the specificity and the sensitivity of sequence detection is able to achieve almost 100%, but conventional PCR-based spoligotyping and MIRU cannot.
  • detection of 192 samples can be completed within 48 hours. Accordingly, advantages of the 25 tagSNPs genotyping method described herein include excellent specificity and sensitivity, less sample requirements, rapid and large scale detection.

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