Molecular technologies used in detecting of sensitive and isoniazid-resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Two variants for the detection of single nucleotide polymorphisms in codon 315 of the katG gene of Mycobacterium tuberculosis (MTB) (mutations in this gene are associated with resistance to isoniazid, which is an antituberculosis drug of the first line) have been developed. Two sets of primers, either of which included an additional competitive blocking primer with a 3′-terminal phosphate group (in order to prevent nonspecific amplification), permitted the identification of the most frequent AGC → ACC and AGC → AGA point mutations in codon 315 of the katG gene. Conduction of PCR with a set of two primers, one of which contained five LNA monomers, permitted the detection of any of the six known mutations in codon 315 of the katG gene and, thereby, for the discrimination between isoniazid-sensitive and isoniazid-resistant MTB. The purity and structure of the 17 bp long primers containing LNA-modified nucleotides were characterized by time-of-flight MALDI mass spectrometry, and the 17 bp duplex formed by two LNA-containing complementary oligonucleotides was analyzed by thermal denaturation. The molecular genetic test systems created for differentiating between the wild-type MTB isolates and isoniazid-resistant MTB (an antituberculosis drug of the first line) can be used in clinical laboratories equipped with standard PCR devices; such systems permit the shortening of the time required for the detection of isoniazid resistance of MTB: from 1–3 months by the standard bacteriological methods to 1–3 days by PCR.     

Identification of wild-type <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isolates and point mutations associated with isoniazid resistance

Isoniazid resistance in Mycobacterium tuberculosis (MBT) is associated with point mutations in codon 315 of the katG gene. Two PCR technique were developed for detection of point mutations in codon 315. Most frequent point mutations (AGC → ACC and AGC → AGA) were identified in codon 315 by using two sets of primers, either of which included an additional competitive blocking primer with a 3′-terminal phosphate group in order to prevent nonspecific amplification. PCR with a set of two primers, one of which contained five locked nucleic acid monomers (LNA), permits one to detect any of six known mutations in codon 315 of katG and, thereby, discriminate between isoniazid-sensitive and resistant MBT isolates. The structure and purity of the 17-nt long LNA-containing oligonucleotides were characterized by MALDI-TOF mass spectrometry; and the 17 bp duplex formed by two LNA-containing complementary oligonucleotides was analyzed by thermal denaturation.     

Characterization of oligonucleotides with LNA-monomers for PCR detection of point mutations in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> genome

Point mutations associated with isoniazid resistance in Mycobacterium tuberculosis (MTB) have been analyzed in codon 315 of the katG gene by conventional polymerase chain reaction (PCR) using primers containing locked nucleic acid (LNA) modified nucleotides. Purity and structure of primers containing 5 LNA monomers of 17 nucleotides in length were characterized by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and a 17-mer duplex formed by two complementary oligonucleotides was characterized by the method of thermal denaturation. The duplex containing five LNA monomers per each strand was characterized by a higher melting temperature than it was expected using extrapolation of theoretical calculation for nucleotide modification of one strand of the duplex. Detection of any of six possible mutations in katG codon 315 (i.e. discrimination between sensitive and resistant MTB) requires just one PCR employing a set of two primers with one LNA-modified primer; this is an important advantage of oligonucleotides containing LNA over unmodified nucleotides: employment of multiplex PCR would require up to 12 primers. Problems of control of oligonucleotide modification by LNA monomers are discussed.     

结核耐药基因的基因芯片检测及临床应用

目的:采用基因芯片技术对结核分枝杆菌中常见耐药基因rpoB、katG及inhA进行检测,以了解结核分枝杆菌的耐药情况,及基因芯片技术检测结核菌耐药基因的临床应用价值。方法:收集40例涂片抗酸染色阳性并经分枝杆菌菌种鉴定芯片鉴定为结核的样本进行结核耐药基因检测。结果:40例样本中,14例无法判读结果,占35%,检出26例,检出率为65%。其中,无突变的野生型21例,占52.5%;突变型5例,总突变率为12.5%;3例rpoB基因的531点单独突变(TCG→TTG),突变率为7.5%;2例katG基因的315点单独突变(AGC→ACC),突变率为5%。结论:结核耐药基因芯片试剂盒检测结核菌耐药基因时针对单个菌落,用痰样本直接检测耐药基因虽能简便快速地了解结核分枝杆菌的耐药情况,但会出现一些无法判读的结果,原因须进一步探讨。     

Barcoding old Korean lepidopteran specimens using newly designed specific primer pairs

Currently, DNA barcodes are often required to be analyzed using old museum specimens when they are the only available specimens for rare or endangered species, or even type series. In this study, using eight universal primers and newly designed 315 species-specific primers, we tried to recover full-length barcode sequences from 45 dried specimens of 36 butterfly species collected between 1959 and 1980 in Korea. The eight universal primers failed entirely in the PCR amplification and sequencing of all the specimens. On the other hand, 284 primer pairs consisting of the 315 primers, targeting fragments of 71–417 bp, amplified various lengths of barcode sequences from all specimens. The fragments were successfully combined to generate the barcode sequences ranging from 444 bp to 658 bp. Notably, of the 284 primer pairs, 26 primer pairs designed for Limenitis camilla, Argynnis niobe, and Brenthis daphne successfully amplified the barcode sequences of congeneric species, Limenitis doerriesi, Argynnis nerippe, and Brenthis ino, suggesting that the species-specific primers can be available for analyzing barcode sequences of closely related species. Our study reveals that the newly designed species-specific primers will be effective in acquiring COI sequences from old butterfly specimens.     

The detection of beta-globin gene mutations in beta-thalassemia using oligonucleotide probes and amplified DNA

DNA amplification combined with the use of synthetic oligonucleotide probes has become an important tool in the identification of base substitutions. We report the use of this DNA amplification technique for the detection of mutations in beta-thalassemia. A series of oligonucleotide primers are synthesized which span the beta-globin gene; one primer is complementary to the coding strand and the other to the non-coding strand. The primers are chosen so that there is little homology with other DNA segments, especially the delta gene. Each set of primers spans an area of the gene between 100 and 300 bp, while the suspected mutation point is located between these two primers. With the use of such a primer set, the beta-globin gene region is amplified by denaturation, annealing and DNA synthesis. The amplification cycle is repeated 25-30 times, using the Klenow fragment of DNA polymerase I. The resulting amplified DNA is hybridized with normal and synthetic deoxynucleotide probes using a standard dot-blot method. We have designed a set of primers and experimental conditions which should prove useful to diagnostic centers for detection of numerous beta-thalassemia mutations.     

结核分枝杆菌katG基因突变的研究

探讨编码过氧化氢-过氧化物酶的katG基因突变与结核分枝杆菌异烟肼(INH)耐药性的相关关系。根据结核分枝杆菌GenBank中的katG序列,自行设计特异性寡聚核苷酸引物,采用聚合酶链反应-单链构象多态性(PCR-SSCP)分析和直接测序法(DS)分析结核分枝杆菌中katG基因突变情况。以HR37Rv标准株为对照。所有23株敏感菌均未有SSCP结果异常;35株耐药菌中,有2株(5．7％)katG基因扩增阴性,且发生在高度耐药菌中。进一步分析发现,SSCP法突变检出23株(65．7％),测序法突变检出24株(68．6％),符合率为95．8％(23／24)。参照测序法对耐药菌突变序列的分析结果,PCR—SSCP敏感、特异,可快速检测结核分枝杆菌katG耐药基因突变,有利于耐药结核分枝杆菌耐药性的快速检测。     

A simple method for genotyping the bovine growth hormone gene

An allele-specific polymerase chain reaction (PCR) amplification method was developed to determine the genotypes at the bovine growth hormone locus that result from two nucleotide substitutions in exon 5 of the gene. This method was a multiplex PCR (ASM–PCR) employing a common primer pair and two allele-specific reverse primers. The common primer pair was designed to amplify a target region containing two substitution points from the three variants of the bovine growth hormone gene. The allele-specific primers were designed to be mismatched with other genotypes at the 3' end of oligonucleotides. When the common and allele-specific reverse primers competed with each other, the shorter allele-specific fragments were amplified preferentially. Consequently, the PCR products of the variant-specific fragments were 347, 483 and 656 bp for alleles A, B and C, respectively, of the bovine growth hormone gene. Genotypes of the bovine growth hormone gene were easily identified by agarose gel electrophoresis of PCR products. The results suggested that this multiplex PCR method would be useful for identification of genetic variants caused by point mutations.     

Mutations linked with antimicrobial resistance in Mycobacterium tuberculosis isolated from tuberculosis patients in the Samara region

A total of 234 M. tuberculosis isolates were used to demonstrate the leading role of mutations in, respectively, codon 531 of gene rpoB (90.0%) and codon 315 of gene katG (92.9%), in the development of resistance to rifampicin and isoniazid by the methods of reverse hybridization with oligonucleotide probes and the sequencing of gene stretches. The levels of primary resistance of M. tuberculosis to rifampicin, isoniazid and multiresistance, according to the molecular-genetic analysis, were 41.0%, 57.7% and 37.2% respectively. The coincidence of the results of the bacteriological and molecular-genetic analyses of the antimicrobial resistance of the isolates was 90.4% and 95.3% for isoniazid and rifampicin respectively. The prevalence of individual types of mutations, linked with antimicrobial resistance, in the presence of a considerable spread of strains of the family Beijing in the region may be indicative of the limited number of M. tuberculosis clones circulating in the region.     

Evaluation of reasons of the MDR M. tuberculosis strains dissemination by analysis of the rifampicin and/or isoniazid resistant isolates

During the last years in Novosibirsk region of Russia the rate of TB patients infected by MDR strains of M. tuberculosis has been constantly increasing. This increase may occur as a result of the spontaneously mutated mycobacterium selection during treatment of patients or as a result of primary infection by the resistant M. tuberculosis, or also, as a result of both reasons in combination. If the main reason of MDR strain dissemination is selection of resistant bacterium during patient treatment, the equal apportionment of the dominated mutation into the mycobacterium genotypes would be observed. If the main reason is the primary infection by resistant M. tuberculosis, the unequal apportionment would be revealed. For deeper understanding of the main reasons of the fast MDR strains spreading in the region, the distribution of the main mutations over genotypes of strains in Novosibirsk (170 isolates) and Tomsk prison (51 isolates) was investigated. Mutations in rpoB gene associated with the rifampicin resistance and in katG (isoniazid resistance) were detected by biochips. M. tuberculosis genotypings were carried out by IS6110 PCR typing or MIRU typing, in the last method the twelve loci (MIRU 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40) have been used. The most frequent mutation in the rpoB gene was Ser531-->Leu (60-70% of the rifampicin resistant strains) and Ser315-->Thr in gene katG (80% of the isoniazid resistant M. tuberculosis). Both in Novosibirsk and in Tomsk prison the rates of clustered cases transmissions were high (69 and 63% respectively). Analysis of the distribution of the dominated mutations Ser531-->Leu (rpoB) and Ser315-->Thr (katG) revealed that all of them were detected in each clusters, but in Novosibirsk there were only two clusters, in which the percentage of strains, containing mutation Ser531-->Leu (rpoB) were higher (85.7% and 77.7% respectively, P < 0.05), then in others. Among the Tomsk prison's clusters it was revealed one in which the proportion of the Ser3 15-->Thr mutation in katGwas higher (96.4%, P < 0.05). The nonuniform distribution of the dominated mutations highlighted that the epidemic spread of drug-resistant strains of M. tuberculosis in region resulted from the selection of them during patient treatment and the subsequent transmission by TB patients.     

A Pyrosequencing assay for rapid recognition of SNPs in Mycobacterium tuberculosis embB306 region

Ethambutol (EMB) is in use worldwide as a first-line anti-tuberculosis drug and substitutions in codon 306 of the embB gene are the most common mutations found in EMB resistant Mycobacterium tuberculosis (MTB) strains. Pyrosequencing is a real time sequencing method able to rapidly detect mutations in a large number of samples. Using this technique we analyzed, in parallel with conventional sequencing, a 24 bp region of the embB gene of 28 MTB clinical isolates. Pyrosequencing efficiently identified all embB306 mutations, detecting three different single-base substitutions leading to 2 amino acid changes (Met to Val or Ile). Mutated embB alleles were detected in 2 multidrug-resistant (MDR) EMB-susceptible strains. Overall, our results demonstrated that the Pyrosequencing method efficiently recognizes mutations in embB in a very short time and represents a valid molecular method to detect mutations in the MTB embB306 region.     

Genotypic and phenotypic characteristics of tunisian isoniazid-resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains

Forty three isoniazid (INH)-resistant Mycobacterium tuberculosis isolates were characterized on the basis of the most common INH associated mutations, katG315 and mabA −15C→T, and phenotypic properties (i.e. MIC of INH, resistance associated pattern, and catalase activity). Typing for resistance mutations was performed by Multiplex Allele-Specific PCR and sequencing reaction. Mutations at either codon were detected in 67.5% of isolates: katG315 in 37.2, mabA −15C→T in 27.9 and both of them in 2.4%, respectively. katG sequencing showed a G insertion at codon 325 detected in 2 strains and leading to amino acid change T326D which has not been previously reported. Distribution of each mutation, among the investigated strains, showed that katG S315T was associated with multiple-drug profile, high-level INH resistance and loss or decreased catalase activity; whereas the mabA −15C→T was more prevalent in mono-INH resistant isolates, but it was not only associated with a low-level INH resistance. It seems that determination of catalase activity aids in the detection of isolates for which MICs are high and could, in conjunction with molecular methods, provide rapid detection of most clinical INH-resistant strains.     

Two recurrent nonsense mutations and a 4 bp deletion in a quasi-symmetric element in exon 37 of the NF1 gene

We screened a total of 92 unrelated patients with neurofibromatosis type 1 (NF1) for mutations in exon 37 of the NF1 gene, by using temperature gradient gel electrophoresis. Two novel mutations were found: a 4 bp deletion in a so-called quasi-symmetric element (6789delTTAC) and a recurrent nonsense mutation, which was identified in two unrelated patients, at codon 2264 (C6792A). The independent origin of the latter mutation in two families was confirmed by haplotype analysis. The nonsense mutation and the 4 bp deletion are both predicted to lead to a truncated protein product lacking the Cterminal 20% (aproximately) of its sequence. The occurrence of three independent mutations among 92 NF1 patients at codons 2263–2264 (exon 37) suggests that a specific search for mutations in this area should be undertaken in screening programs for NF1 mutations.     

The use of macroarrays for the identification of MDR Mycobacterium tuberculosis

The emergence of Mycobacterium tuberculosis (Mtb), resistant to both isoniazid (INH) and rifampicin (RIF) (MDR-TB), is an increasing threat to tuberculosis control programs. Susceptibility testing of Mtb complex isolates by phenotypic methods requires a minimum of 14 days from a primary specimen. This can be reduced significantly if molecular analysis is used. Low density oligonucleotide arrays (macroarrays) have been used successfully for the detection of RIF resistance in Mtb. We describe the use of macroarray technology to identify Mtb complex isolates resistant to INH and/or RIF. The macroarray MDR-Mtb screen has been designed to detect mutations in the RIF resistance determining region (RRDR) of Mtb rpoB and loci in katG and mabA-inhA associated with INH resistance. A panel of Mtb isolates containing 38 different RRDR genotypes, 4 different genotypes within codon 315 of katG and 2 genotypes at mabA-inhA was used to validate the macroarray. The wild type (WT) genotype was correctly identified at all three loci. Of the 37 mutant rpoB genotypes, 36 were correctly detected; the single mutant not detected contained a 9 base insertion. All mutations within katG and mabA-inhA were correctly identified. We conclude that this low cost, rapid system can usefully detect the mutations associated with the vast majority of MDR-Mtb.     

Prevalence of mutations at codon 463 of katG gene in MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus and application of the method in rapid diagnosis

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. In respect to high GC content of Mycobacterium tuberculosis, nonsynonymous mutations are dominant in this group. In this study a collection of 145 M. tuberculosis isolates was used to evaluate the conferring mutations in nucleotide 1388 of katG gene (KatG463) in resistance to isoniazid. A PCR-RFLP method was applied in comparison with DNA sequencing and anti-mycobacterial susceptibility testing. From all studied patients, 98 (67.6%) were men, 47 (32.4%) were women, 3% were <15 and 9% were >65 years old; male to female ratio was 1:2.4. PCR result of katG for a 620-bp amplicon was successful for all purified M. tuberculosis isolates and there was no positive M. tuberculosis culture with PCR negative results (100% specificity). Subsequent PCR RFLP of the katG identified mutation at KatG463 in 33.3%, 57.8% and 59.2% of our clinically susceptible, multidrug resistant TB (MDR) and extensively drug resistant (XDR) isolates, respectively. Strains of H37Rv and Academic had no any mutations in this codon. M. bovis was used as a positive control for mutation in KatG463. Automated DNA sequencing of the katG amplicon from randomly selected INH-susceptible and resistant isolates verified 100% sequence accuracy of the point mutations detected by PCR-RFLP. We concluded that codon 463 was a polymorphic site that is associated to INH resistance (a missense or "quiet" mutation). RFLP results of katG amplicons were identical to those of sequence method. Our PCR-RFLP method has a potential application for rapid diagnosis of M. tuberculosis with a high specificity.     

Complete nucleotide sequence and organization of the mitochondrial genome of eri-silkworm,Samia cynthia ricini (Lepidoptera: Saturniidae)

Samia cynthia ricini is a commercial silk-producing insect that is now reared year-round in Korea, with the expectation of being utilized for diverse purposes. In this report, we present the complete mitochondrial genome (mitogenome) of S. c. ricini. The 15,384-bp long S. cynthia ricini mitogenome was amplified into 26 short fragments using three long overlapping fragments using primers designed from reported lepidopteran mitogenome sequences. The genome comprises 37 genes (13 protein-coding genes, two rRNA genes, and 22 tRNA genes), and one large non-coding region termed the A + T-rich region. The A/T content of the third codon position was 91.7%, which was 18.8% and 21.6% higher than those of first and second codon positions, respectively. The high A/T content in the genome is reflected in codon usage, accounting for 39.5% of A/T-composed codons (TTA, ATT, TTT, and ATA). Unlike a previous report on the start codon for the COI gene, the S. c. ricini COI gene commences with a typical ATT codon. A total of 221 bp of non-coding sequences are dispersed in 17 regions, ranging in size from 1 to 54 bp, which comprise 1.4% of the total genome. One of the non-coding sequence located between tRNA^Gln and ND2 (54 bp) has 77% sequence homology with the 5′-sequence of the neighboring ND2 gene, suggesting partial duplication of the sequence during evolution. The 361-bp long A + T-rich region contains an 18 bp-long poly-T stretch, ATAGA motif, ATTTA element, microsatellite-like A/T sequence, poly-A stretch and one tRNA-like sequence, as typically found in Lepidoptera including Bombycoidea.     

Microarray and allele specific PCR detection of point mutations in Mycobacterium tuberculosis genes associated with drug resistance

Global public health is threatened by the emergence of potentially dangerous antibiotic drug-resistant strains of Mycobacterium tuberculosis. Point mutations in certain M. tuberculosis genes are associated with the resistance of M. tuberculosis strains to antibiotic drugs. The purpose of this study was to develop a suitable microarray-based protocol for the detection of point mutations in M. tuberculosis genes associated with drug resistance. We initially developed a conventional, oligonucleotide microarray protocol and used it to detect and identify on a single microarray slide a number of point mutation-containing rpoB and katG gene target sequences. However, the occurrence of some non-specific hybridization led us to the development of an improved protocol based on allele specific PCR combined with tags/anti-tags and microarrays. This protocol was evaluated by detecting point mutations in M. tuberculosis katG and rpoB gene templates produced by recombinant PCR. The methodology allowed sequences containing single point mutations to be readily distinguished from wild type sequences. The data obtained with the improved protocol had strong and specific signals and relatively low amounts of non-specific hybridization. We successfully used this protocol to detect and identify (<8 h) a number of clinically relevant point mutations in the rpoB, katG and rpsL genes of M. tuberculosis clinical isolates. Our allele specific PCR/tags and anti-tags/microarray protocol has several advantages over our conventional oligonucleotide microarray protocol, and it may have broad applications for point mutation detection.     

Mutation analysis of codons 345 and 347 of rhodopsin gene in Indian retinitis pigmentosa patients

More than 100 mutations have been reported till date in the rhodopsin gene in patients with retinitis pigmentosa. The present study was undertaken to detect the reported rhodopsin gene point mutations in Indian retinitis pigmentosa patients. We looked for presence or absence of codon 345 and 347 mutations in exon 5 of the gene using the technique of allele-specific polymerase chain reaction by designing primers for each mutation. We have examined 100 patients from 76 families irrespective of genetic categories. Surprisingly, in our sample the very widely reported highly frequent mutations of codon 347 (P → S/A/R/Q/L/T) were absent while the codon 345 mutation V → M was seen in three cases in one family (autosomal dominant form) and in one sporadic case (total two families). This is the first report on codon 345 and 347 mutation in Indian retinitis pigmentosa subjects.     

Detection of mutations associated with multidrug-resistant Mycobacterium tuberculosis clinical isolates

Antimicrobial resistance was studied in 100 Mycobacterium tuberculosis strains selected randomly from sputum cultures of newly diagnosed tuberculosis patients. Resistance of the isolates to rifampicin, isoniazid, and ethambutol was tested by both drug susceptibility testing (DST) and allele-specific PCR (AS-PCR). A total of 19 (19%) isolates were found resistant to at least one of the antituberculosis drugs investigated by PCR compared with 14 (14%) resistant isolates detected by DST. Eleven mutations were detected by AS-PCR in the rpoB gene (codons 516, 526, and 531), associated with rifampicin resistance, a marker of multidrug-resistant tuberculosis (MDR-TB), 14 mutations in the katG gene codon 315 that confers resistance to isoniazid, and nine mutations in the embB gene codon 306 that confers resistance to ethambutol. Mutations in the six multidrug-resistant isolates were confirmed by DNA sequencing. Results were compared with phenotypic DST data. Nineteen different mutation types to at least one of the drugs were found; six isolates (6%) were classified as MDR-TB, defined as resistance to at least rifampicin and isoniazid. The rates of concordance of the PCR with the phenotypic susceptibility test were 71.4, 54.5, and 44.4 for isoniazid, rifampicin, and ethambutol, respectively. These results highlight the importance of molecular epidemiology studies of tuberculosis in understudied regions with a tuberculosis burden to uncover the true prevalence of the MDR-TB.     

Sequence variation of chalcone synthase gene in a spontaneous white-flower mutant of Chinese cabbage-pak-choi

A spontaneous white-flower mutant of Chinese cabbage-pak-choi (Brassica campestris ssp. chinenesis, syn. B. rapa ssp. chinenesis) was found in our test fields, and all the plant characters except flower color were identical with wild type ones. We hypothesized that a mutational event had occurred in the gene coding for chalcone synthase (CHS), the key enzyme of flavonoid biosynthesis pathway. Two genes, later designated BcCHS and BcCHS-wf, were isolated from wild type and mutant Chinese cabbage-pak-choi, respectively, using gene-specific primer pairs. Comparison of the genomic sequences revealed two mutations in BcCHS-wf, both with A to G transitions, one at position +37 bp and the other at +970 bp. Both nucleotide substitutions occurred in AGA codes for arginine into GGA for glycin at residue +13 and into AGC coding for serine at residue +229, respectively. Homologous genes of BcCHS were isolated from another four cruciferous plants, though there were some differences among the genomic and deduced amino acid sequences, the mutation locus of the mutant, as we called it, were identical to the wild type Chinese cabbage-pak-choi.