Patent Grant
11293067
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “2018-03-14-Sequence-Listing” created on Mar. 14, 2018 and is 44,366 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
The present invention relates to the method for detecting Mycobacterium tuberculosis, and more particularly for genotyping M. tuberculosis.
Tuberculosis (TB) 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.
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. Currently, there are two genotyping methods that are commonly used to study tuberculosis transmission (van Deutekom H. et al., J Clin Microbiol 2005, 43(9):4473-4479). 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. The portable data format facilities easy inter-laboratory comparison. To date published, freely accessible databases for strain lineage identification have been developed on the basis of spoligotype signature matching (Brudey K et al., BMC Microbiol 2006, 6:23). Another molecular technique for strain typing of MTB is based on variable number tandem repeats (VNTRs) of mycobacterial interspersed repetitive units (MIRUs) (Mazars E. et al., Proc Natl Acad Sci USA 2001, 98(4):1901-1906; Comas I. et al., PLoS One 2009, 4(11): e7815; Supply P. et al., Mol Microbiol 2000, 36(3):762-771). This method is based on the number of repeats observed at each of the 12, 15 or 24 selected MIRU loci, determined using a PCR-based method.
However, the conventional methods for genotyping MTB have disadvantages including the requirement of large amount of DN sample, time-consumption, insufficient sensitivity and specificity, inability for genotyping particular strains. Therefore, there is a need for the improved method for genotyping MTB.
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. 85, “A” at position 301 of SEQ ID No. 86, “A” at position 301 of SEQ ID No. 87, “G” at position 301 of SEQ ID No. 88, “A” at position 301 of SEQ ID No. 89, “G” at position 301 of SEQ ID No. 90, “G” at position 301 of SEQ ID No. 91, “A” at position 301 of SEQ ID No. 92, “C” at position 301 of SEQ ID No. 93, “C” at position 301 of SEQ ID No. 94, “T” at position 301 of SEQ ID No. 95, “T” at position 301 of SEQ ID No. 96, “T” at position 301 of SEQ ID No. 97, “T” at position 301 of SEQ ID No. 98, “T” at position 301 of SEQ ID No. 99, and “C” at position 301 of SEQ ID No. 100.
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.
In other embodiments, 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.
In the present application, 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:
The primer set can be applied in polymerase chain reaction to amplify a DNA fragment containing a single-nucleotide polymorphism (SNP) of M. tuberculosis. The above primer sets can be used alone or in combination. In some embodiments, the combination of the primer sets can be applied simultaneously in one test tube for PCR test. In some embodiments, any combination selected from the primer sets 1-12 can be applied simultaneously. In some embodiments, any combination selected from the primer sets 13-22 can be applied simultaneously. In some embodiments, any combination selected from the primer sets 23-25 can be applied simultaneously.
In the present application, the 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:
In one embodiment, 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. 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.
In the present application, 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. 85, “A” at position 301 of SEQ ID No. 86, “A” at position 301 of SEQ ID No. 87, “G” at position 301 of SEQ ID No. 88, “A” at position 301 of SEQ ID No. 89, “G” at position 301 of SEQ ID No. 90, “G” at position 301 of SEQ ID No. 91, “A” at position 301 of SEQ ID No. 92, “C” at position 301 of SEQ ID No. 93, “C” at position 301 of SEQ ID No. 94, “T” at position 301 of SEQ ID No. 95, “T” at position 301 of SEQ ID No. 96, “T” at position 301 of SEQ ID No. 97, “T” at position 301 of SEQ ID No. 98, “T” at position 301 of SEQ ID No. 99, and “C” at position 301 of SEQ ID No.100. The detail sequence information are shown as follows.
The above 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, “A” at position 3078579 of genome of the reference strain, “C” at position 3157993 of genome of the reference strain, “C” at position 3426415 of genome of the reference strain, “T” at position 3734189 of genome of the reference strain, “T” at position 3797876 of genome of the reference strain, “T” at position 3859376 of genome of the reference strain, “T” at position 4061113 of genome of the reference strain, “T” at position 4221423 of genome of the reference strain, and “C” at position 4352162 of genome of the reference strain, respectively.
In the present application, the reference strain is M. tuberculosis H37Rv having a complete genome sequence NC_000962.2. Said genome sequence is public information, which is published in the database of National Center for Biotechnology Information (NCBI) entitled “gi57116681/ref. NC_000962.21 Mycobacterium tuberculosis H37Rv chromosome, complete genome”.
Table 1 shows the detail information of the SNP markers of the present application. In Table 1, 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, and the variant allele is the SNP marker of the present application.
In some embodiments, various genotypes of M. tuberculosis possess various combinations of the SNP markers. Preferably, the combination of the SNP markers contains at least two markers, such as 3, 5, 7, 10, 15, 20, or 25 markers. However, in some embodiments, 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.
In some embodiments, the method can further comprises analyzing the mass spectrometry data based on the single-nucleotide polymorphism markers selected from Table 1.
In one preferred embodiment, 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. In some embodiments, 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.
To investigate the transmission, virulence, antimicrobial resistance and other attributes of the MTB, 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.
Materials and Methods
Bacterial Strains and Molecular Typing
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.
Genome Sequencing of MTB Strains
Six MTB strains, W6, M3, M7, A27, A18 and M24, belong to the genogroups modern Beijing, Haarlem, Latin-American Mediterranean (LAM), T, East African-Indian (EAI), and ancient Beijing, respectively. They represent the major types of clinical strains isolated from three different ethnic groups in Taiwan and were taken to whole genome sequencing using the 454 pyro-sequencing approach (Margulies M et al., Nature 2005, 437(7057):376-380). 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.
DNA libraries of six MTB Haarlem and six T clinical isolates were prepared using Nextera DNA sample preparation kit (Illumina, Calif., 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%.
Mapping to the Reference Genome H37Rv
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. 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.
Selection of Strain-Specific SNPs
Based on the result which contains “High-Confidence” differences with at least three non-duplicate reads that (a) show the differences, (b) have at least five bases on both sides of the difference, (c) have few other isolated sequence differences in the read, and (d) have at least one aligned in the forward direction and at least one aligned in the reverse direction. Besides, only those variation sites that all six strains have at least three reads covered and the variation rate larger or equal to 80% were considered as valid. Home-made scripts were used to merge the mapping results of all six strains and parse those valid differences into a MySQL database for further analysis. 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.
For SNP calling of Illumina HiSeq2000 sequence data, mapped sequence data of each sample was analyzed using CLC Genomics Workbench software (Aarhus, Denmark) with default parameters. We applied an additional filter to identify highly reliable SNPs with more than 30-fold depth and >95% variant frequency.
SNP Genotyping Based on the MassArray System
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. In the primer extension procedure, 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. After analyzing the genotype profiles, 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.
Linkage Disequilibrium and Phylogenetic Analysis
Based on the haploview software, the Lewontin D′ measure was used to estimate the intermarker coefficient of linkage disequilibrium (LD) as shown in
Results
Genome Sequencing of Six MTB Clinical Isolates
The overall scheme for selecting lineage-specific DNA markers is shown in a flowchart (
The 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 large contig (>=1,000 bp) numbers were 134˜158 for the three isolates sequenced by 454 GS20 and 196˜200 for the three isolates sequenced by 454 FLX, indicating that large contigs ratio were higher for the three isolates sequenced by 454 FLX. 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.
Sdepth = totalBases/length of H37Rv genome
Genetic Variations of the MTB Clinical Isolates
We totally extracted 9,003 high-confidence (HC) variations (for the definition of HC variants, please see the Method section), including SNPs, multiple nucleotide polymorphisms (MNPs), insertions and deletions (INDELs), from the mapping results of the six isolates. After sorting these variations with reference positions, and at least one isolate with over 80% of variation frequency, there are 3,819 reference positions that all the six isolates got at least three reads covered. For simplicity, 3,582 reference positions contained only SNPs were chose for the following analysis (other 27 positions were INDELs and 210 positions were MNPs).
Among these 3,582 SNPs, 404 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). For those SNPs in coding regions, non-synonymous SNPs seem to have more or equal number them synonymous SNPs, except in M7 isolate. And as we know that the presence of the two novel gene families 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.
SNP Genotyping on 156 M. tuberculosis Clinical Isolates
In order to characterize SNPs in 156 clinical isolates for phylogenetic analysis, 120 lineage-specific SNPs with high confidence scores 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). Five of 120 SNPs were not designable in Sequenom matrix-assisted laser desorption inoization-time of flight mass spectrometry (MALDI-TOF) systems because of high GC contents and/or primer dimmers. 115 of 120 SNPs were designed into 10 multiplex reactions, and were genotyped in 156 clinical M. tuberculosis isolates. We excluded five SNPs with low call rate (<95%) and bad clustering pattern, and the remaining 110 SNPs are used in the following analysis. The false-positive and false-negative rates were both 0% when comparing Sequenom and 454 sequencing data, and the average call rate of each of 110 SNPs in 156 samples were 97%. There were strong correlations between these SNPs in the MTB genomes based on linkage disequilibrium analysis as shown in
Phylogenetic and Grouping Analysis of MTB Isolates
To trace the relationships between 156 clinical isolates, phylogenetic trees were constructed based on 110-SNP or 25-tagSNP information as shown in
Combination of spoligotyping and SNP genotyping data, we characterized the allele frequencies these 110 SNPs in 51 modern Beijing, 25 Haarlem, 11 EAI, 10 ancient Beijing, 7 T and 3 LAM isolates as shown in
32 of 107 (30%) spoligotype-classified isolates were poorly classified using these 25 tagSNPs, and these isolates all belong to Euro American lineage (25 and 7 were classified as Haarlem and T strains based on spoligotype data, respectively). We hypothesized that there are high genetic heterozygosities of spoligotype-classified within Haarlem or T strains, resulting there was no leaf for Haarlem and T strains of decision tree (
Genotyping of MTB by Using 25 tagSNPs
As described above, 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
Discussion
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 transferrable in a format that can be easily shared between laboratories. In this study, we have designed a selection scheme of lineage-specific markers by genome sequencing, comparative analysis, and genotyping with DNA mass spectrometry, and also demonstrated the utility and accuracy of this new typing protocol. Because of its speed and ease of laboratory operation and the simple data format for exchange and comparison, the protocol reported here has the potential to become a new standard method. It should prove valuable for the development of an effective infection-control policy.
Although spoligotyping analysis is a straightforward technique, it is less discriminatory than IS6110 RFLP. Moreover, it is a labor-intensive and time-consuming procedure. Even through strain classification based on spoligotyping can assign MTBC strains to the correct phylogenetic lineages in about 90% of the cases, some strains cannot be classified at all, and others might be misclassified as shown in this study (
Additional genotyping of M. tuberculosis isolates is 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.
The proposed workflow of selecting lineage-specific DNA marker (
While the present invention is disclosed by reference to the preferred embodiments and examples detailed above, it is to be understood that these examples are intended in an illustrative rather than in a limiting sense. It is contemplated that modifications and combinations will readily occur to those skilled in the art, which modifications and combinations will be within the spirit of the invention and the scope of the following claims and its equivalent systems and methods.
This application is a continuation of co-pending U.S. application Ser. No. 14/089,990, filed on Nov. 26, 2013, for which priority is claimed under 35 U.S.C. § 120; and this application claims priority of U.S. Provisional Application No. 61/730,033 filed on Nov. 26, 2012 under 35 U.S.C. § 119(e), the entire contents of all of which are hereby incorporated by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6294328 | Fleischmann et al. | Sep 2001 | B1 |
| 20090285847 | Felgner et al. | Nov 2009 | A1 |
| 20110105531 | Massire | May 2011 | A1 |
| Entry |
|---|
| Lowe et al., “A computer program for selection of oligonucleotide primers chain reactions,” Nucleic Acids research, vol. 18(7), 1990, pp. 1757-1761. |
| International Search Report and Written Opinion, PCT/US2013/071819, dated May 23, 2014, 12 pages. |
| Genbank, NCBI Reference Sequencde: NC_000962.2 “Mycobacterium tuberculosis H37Rv chromosome, complete genome,” [online], dated Aug. 20, 2012. |
| Brudey, K., et al., “Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology,” BMC Microbiology, (2006), vol. 6, No. 23, 17 pages. |
| Van Deutekom, H., et al., “Molecular Typing of Mycobacterium tuberculosis by Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Analysis, a More Accurate Method for Identifying Epidemiological Links between Patients with Tuberculosis,” Journal of Clinical Microbiology, (Sep. 2005), vol. 43, No. 9, pp. 4473-4479. |
| Supply, P., et al., “Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome,” Molecular Microbiology (2000) vol. 36, No. 3, p. 762-771. |
| Comas, I., et al., “Genotyping of Genetically Monomorphic Bacteria: DNA Sequencing in Mycobacterium tuberculosis Highlights the Limitations of Current Methodologies,” PLOS One, (Nov. 12, 2009), 11 pages. |
| Mazars, E., et al., “High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology,” PNAS, (Feb. 13, 2001), vol. 98, No. 4, pp. 1901-1906. |
| Dou, H-Y, et al., “Utility and evaluation of new variable-Number tandem-repeat systems for genotyping Mycobacterial tuberculosis isolates,” Journal of Microbiological Methods, (2009), vol. 77, pp. 127-129. |
| Dou, H-Y, et al., “Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosis in Taipei,” BMC Infectious Diseases, (2008), vol. 8, No. 170, 12 pages. |
| Chang, J-R., et al., “Genotypic analysis of genes associated with transmission and drug resistance in the Beijing lineage of Mycobacterium tuberculosis,” Clin. Microbiol. Infec. (2011), vol. 17, pp. 1391-1396. |
| Dou, H-Y., et al., “Associations of Mycobacterium tuberculosis genotypes with different ethnic and migratory populations in Taiwan,” Infection, Genetics and Evolution, vol. 8, (2008), pp. 323-330. |
| Margulies, M., et al., “Genome sequencing in microfabricated high-density picolitre reactors,” Nature, vol. 437, (Sep. 25, 2005), 6 pages. |
| Bouakaze, C., et al., “Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry-Based Single Nucleotide Polymorphism Genotyping Assay Using iPLEX Gold Technology for Identification of Mycobacterium tuberculosis Complex Species and Lineages,” Journal of Clinical Microbiology, (Sep. 2011), vol. 49, No. 9, pp. 3292-3299. |
| Invitation to Pay Additional Fees, PCT/US2013/071819, dated Mar. 19, 2014, 2 pages. |
| Number | Date | Country | |
|---|---|---|---|
| 20180208974 A1 | Jul 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61730033 | Nov 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14089990 | Nov 2013 | US |
| Child | 15921323 | US |
