Rapid identification and detection of pine pathogenic fungi associated with mountain pine beetles by padlock probes

Fifteen million hectares of pine forests in western Canada have been attacked by the mountain pine beetle (Dendroctonus ponderosae; MPB), leading to devastating economic losses. Grosmannia clavigera and Leptographium longiclavatum, are two fungi intimately associated with the beetles, and are crucial components of the epidemic. To detect and discriminate these two closely related pathogens, we utilized a method based on ligase-mediated nucleotide discrimination with padlock probe technology, and signal amplification by hyperbranched rolling circle amplification (HRCA). Two padlock probes were designed to target species-specific single nucleotide polymorphisms (SNPs) located at the inter-generic spacer 2 region and large subunit of the rRNA respectively, which allows discrimination between the two species. Thirty-four strains of G. clavigera and twenty-five strains of L. longiclavatum representing a broad geographic origin were tested with this assay. The HRCA results were largely in agreement with the conventional identification based on morphology or DNA-based methods. Both probes can also efficiently distinguish the two MPB-associated fungi from other fungi in the MPB, as well as other related fungi in the order Ophiostomatales. We also tested this diagnostic method for the direct detection of these fungi from the DNA of MPB. A nested PCR approach was used to enrich amplicons for signal detection. The results confirmed the presence of these two fungi in MPB. Thus, the padlock probe assay coupled with HRCA is a rapid, sensitive and reproducible method for the identification and detection of these ophiostomatoid fungi.     

Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a “probe dropout” manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.     

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ.     

One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates

In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43.     

Rapid Identification of Seven Waterborne <Emphasis Type="Italic">Exophiala</Emphasis> Species by RCA DNA Padlock Probes

The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37–40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe–template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.     

A duplex real-time PCR assay for detection of mutT4 and mutT2 mutations in Mycobacterium tuberculosis of Beijing genotype

To determine the polymorphism of mutT genes of Mycobacterium tuberculosis of Beijing genotype, we developed a duplex real-time PCR assay based on hybridization probes for the Roche LightCycler instrument. The assay rapidly detects mutations at codons 48 and 58 of genes mutT4 and at mutT2, respectively.     

Nanodiagnostic Method for Colorimetric Detection of Mycobacterium tuberculosis 16S rRNA

A nanodiagnostic method using nucleic acid sequence-based amplification (NASBA) and gold nanoparticle probes (AuNP probes) was developed for colorimetric detection of Mycobacterium tuberculosis. The primers targeting 16S rRNA were used for the amplification of mycobacterial RNA by the isothermal NASBA process. The amplicons were hybridized with specific gold nanoparticle probes. The RNA–DNA hybrids were colorimetrically detected by the accumulation of gold nanoparticles. Using this method, 10 CFU ml^?1 of M. tuberculosis was detected within less than 1 h. Results obtained from the clinical specimens showed 94.7% and 96% sensitivity and specificity, respectively. No interference was encountered in the amplification and detection of M. tuberculosis in the presence of non-target bacteria, confirming the specificity of the method.     

Development of a single multiplex amplification refractory mutation system PCR for the detection of rifampin-resistant Mycobacterium tuberculosis

A rapid and simple method for the detection of drug-resistant Mycobacterium tuberculosis is critical for the efficient treatment and control of this pathogen in developing country. Here we developed a single multiplex amplification refractory mutation system (M-ARMS) PCR, in which chimeric-primer and temperature switch PCR (TSP) strategy were included. Using this method, we detected rifampin resistance-associated mutations at codons 511, 516, 526 and 531 in the rifampin resistance-determining region of rpoB gene. The performance of M-ARMS-PCR assay was evaluated with 135 cultured isolates of M. tuberculosis. The sensitivity and specificity were 94.2% and 100%, respectively, compared with direct DNA sequencing, and 86.67% and 89.71%, respectively, compared with culture-based phenotypic drug susceptibility testing. Therefore, this newly-developed M-ARMS-PCR method is useful and efficient with an intended application in provincial Centers for Disease Control and Prevention for rapid detection of rifampin resistance-associated mutations.     

Detection of inhibitors of phenotypically drug-tolerant Mycobacterium tuberculosis using an in vitro bactericidal screen

Many whole cell screens of chemical libraries currently in use are based on inhibition of bacterial growth. The goal of this study was to develop a chemical library screening model that enabled detection of compounds that are active against drug-tolerant non-growing cultures of Mycobacterium tuberculosis. An in vitro model of low metabolically active mycobacteria was established with 8 and 30 day old cultures of M. smegmatis and M. tuberculosis, respectively. Reduction of resazurin was used as a measure of viability and the assay was applied in screens of chemical libraries for bactericidal compounds. The model provided cells that were phenotypically-resilient to killing by first and second-line clinical drugs including rifampicin. Screening against chemical libraries identified proteasome inhibitors, NSC310551 and NSC321206, and a structurally-related series of thiosemicarbazones, as having potent killing activity towards aged cultures. The inhibitors were confirmed as active against virulent M. tuberculosis strains including multi- and extensively-drug resistant clinical isolates. Our library screen enabled detection of compounds with a potent level of bactericidal activity towards phenotypically drug-tolerant cultures of M. tuberculosis.     

PCR-linked reverse DNA hybridization using oligonucleotide-specific probes of rpoB for identification of Mycobacterium avium and Mycobacterium intracellulare

A PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes (Avp and Intp) of Mycobacterium avium and Mycobacterium intracellulare, respectively, were evaluated for the differentiation and identification of M. avium and M. intracellulare culture isolates. Among the 504 culture isolates tested by this method, 48 strains showed positive results for M. avium and 60 strains showed positive results for M. intracellulare. The other 396 culture isolates showed negative results for both M. avium and M. intracellulare. These results were consistent with those obtained from partial rpoB (306 bp) sequence analysis and biochemical tests. The negative strains obtained by this DNA hybridization method were identified as M. tuberculosis (366 strains), M. peregrinum (11 strains), M. abscessus (9 strains), M. fortuitum (8 strains), and M. flavescens (2 strains) by rpoB DNA sequence analysis. Due to the high sensitive and specific result obtained by this assay, we suggest that this PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes of M. avium and M. intracellulare would be used for the rapid and precise method for differentiation and identification of M. avium and M. intracellulare.     

Multiplex allele-specific PCR combined with PCR-RFLP analysis for rapid detection of gyrA gene fluoroquinolone resistance mutations in Mycobacterium tuberculosis

A combined use of MAS-PCR (multiplex allele-specific PCR) and PCR-RFLP (PCR restriction fragment length polymorphism), was established to detect mutations in codons 90, 91 and 94 of the gyrA gene in Mycobacterium tuberculosis (M. tuberculosis). With conventional phenotypic drug susceptibility testing as a reference standard, the sensitivity, specificity and accuracy of the modified method for gyrA gene mutation detection were 70.8%, 100% and 84.8% respectively.     

Detection and Enumeration of Methanotrophs in Acidic Sphagnum Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for Methylocella palustris

Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 10⁶ cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 10⁴ type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10⁶ type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (±0.2) × 10⁶ cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.     

Frequency and Geographic Distribution of gyrA and gyrB Mutations Associated with Fluoroquinolone Resistance in Clinical Mycobacterium Tuberculosis Isolates: A Systematic Review

Background

The detection of mutations in the gyrA and gyrB genes in the Mycobacterium tuberculosis genome that have been demonstrated to confer phenotypic resistance to fluoroquinolones is the most promising technology for rapid diagnosis of fluoroquinolone resistance.

Methods

In order to characterize the diversity and frequency of gyrA and gyrB mutations and to describe the global distribution of these mutations, we conducted a systematic review, from May 1996 to April 2013, of all published studies evaluating Mycobacterium tuberculosis mutations associated with resistance to fluoroquinolones. The overall goal of the study was to determine the potential utility and reliability of these mutations as diagnostic markers to detect phenotypic fluoroquinolone resistance in Mycobacterium tuberculosis and to describe their geographic distribution.

Results

Forty-six studies, covering four continents and 18 countries, provided mutation data for 3,846 unique clinical isolates with phenotypic resistance profiles to fluoroquinolones. The gyrA mutations occurring most frequently in fluoroquinolone-resistant isolates, ranged from 21–32% for D94G and 13–20% for A90V, by drug. Eighty seven percent of all strains that were phenotypically resistant to moxifloxacin and 83% of ofloxacin resistant isolates contained mutations in gyrA. Additionally we found that 83% and 80% of moxifloxacin and ofloxacin resistant strains respectively, were observed to have mutations in the gyrA codons interrogated by the existing MTBDRsl line probe assay. In China and Russia, 83% and 84% of fluoroquinolone resistant strains respectively, were observed to have gyrA mutations in the gene regions covered by the MTBDRsl assay.

Conclusions

Molecular diagnostics, specifically the Genotype MTBDRsl assay, focusing on codons 88–94 should have moderate to high sensitivity in most countries. While we did observe geographic differences in the frequencies of single gyrA mutations across countries, molecular diagnostics based on detection of all gyrA mutations demonstrated to confer resistance should have broad and global utility.     

Rapid detection of Mycobacterium tuberculosis in clinical samples by multiplex polymerase chain reaction (mPCR)

Although the multi-copy and specific element IS6110 provides a good target for the detection of Mycobacterium tuberculosis complex by PCR techniques, the emergence of IS6110-negative strains suggested that false negative may occur if IS6110 alone is used as the target for detection. In this report, a multiplex polymerase chain reaction (mPCR) system was developed using primers derived from the insertion sequence IS6110 and an IS-like elements designated as B9 (GenBank accession no. U78639.1) to overcome the problem of detecting negative or low copy IS6110 containing strains of M. tuberculosis. The mPCR was evaluated using 346 clinical samples which included 283 sputum, 19 bronchial wash, 18 pleural fluid, 9 urine, 7 CSF, 6 pus, and 4 gastric lavage samples. Our results showed that the sensitivity (93.1 %) and specificity (89.6 %) of the mPCR system exceeds that of the conventional method of microscopy and culture. The mPCR assay provides an efficient strategy to detect and identify M. tuberculosis from clinical samples and enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.     

Use of Rolling Circle Amplification to Rapidly Identify Species of Cladophialophora Potentially Causing Human Infection

The genus Cladophialophora comprises etiologic agents of disease in immunocompetent patients, ranging from mild cutaneous colonization to cerebral encephalitis, in addition to saprobic species. Due to the high degree of phenotypic similarity between closely related species of the genus, identification problems are imminent. In the present study, we described rapid and sensitive rolling circle amplification (RCA) method based on species-specific padlock probes targeted for the internal transcribed spacer regions of rDNA. ITS regions of 12 Cladophialophora species were sequenced, and subsequently, 10 specific padlock probes were designed for the detection of single nucleotide polymorphisms. The majority of circularizable padlock probes were designed based on single nucleotide polymorphisms (SNPs), while for C. bantiana, C. immunda and C. devriesii were characterized by two or more nucleotides. Individual species-specific probes correctly identified in all ten Cladophialophora species correctly by visualization on 1.2 % agarose gels used to verify specificity of probe-template binding; no cross-reactivity was observed. Simplicity, sensitivity, robustness and low costs provide RCA a distinct place among isothermal techniques for DNA diagnostics. However, restriction and specificity and sensitivity should be lowered and increased, respectively, to be useful for a wide variety of clinical applications.     

Comparative Evaluation of Sloppy Molecular Beacon and Dual-Labeled Probe Melting Temperature Assays to Identify Mutations in Mycobacterium tuberculosis Resulting in Rifampin,Fluoroquinolone and Aminoglycoside Resistance

Several molecular assays to detect resistance to Rifampin, the Fluoroquinolones, and Aminoglycosides in Mycobacterium tuberculosis (M. tuberculosis) have been recently described. A systematic approach for comparing these assays in the laboratory is needed in order to determine the relative advantage of each assay and to decide which ones should be advanced to evaluation. We performed an analytic comparison of a Sloppy Molecular Beacon (SMB) melting temperature (Tm) assay and a Dual labeled probe (DLP) Tm assay. Both assays targeted the M. tuberculosis rpoB, gyrA, rrs genes and the eis promoter region. The sensitivity and specificity to detect mutations, analytic limit of detection (LOD) and the detection of heteroresistance were tested using a panel of 56 clinical DNA samples from drug resistant M. tuberculosis strains. Both SMB and DLP assays detected 29/29 (100%) samples with rpoB RRDR mutations and 3/3 (100%) samples with eis promoter mutations correctly. The SMB assay detected all 17/17 gyrA mutants and 22/22 rrs mutants, while the DLP assay detected 16/17 (94%) gyrA mutants and 12/22 (55%) rrs mutants. Both assays showed comparable LODs for detecting rpoB and eis mutations; however, the SMB assay LODs were at least two logs better for detecting wild type and mutants in gyrA and rrs targets. The SMB assay was also moderately better at detecting heteroresistance. In summary, both assays appeared to be promising methods to detect drug resistance associated mutations in M. tuberculosis; however, the relative advantage of each assay varied under each test condition.     

PCR-generated padlock probes detect single nucleotide variation in genomic DNA

Circularizing oligonucleotide probes, so-called padlock probes, have properties that should prove valuable in a wide range of genetic investigations, including in situ analyses, genotyping and measurement of gene expression. However, padlock probes can be difficult to obtain by standard oligonucleotide synthesis because they are relatively long and require intact 5′- and 3′-end sequences to function. We describe a PCR-based protocol for flexible small-scale enzymatic synthesis of such probes. The protocol also offers the advantage over chemical synthesis that longer probes can be made that are densely labeled with detectable functions, resulting in an increased detection signal. The utility of probes synthesized according to this protocol is demonstrated for the analysis of single nucleotide variations in human genomic DNA both in situ and in solution.     

Development of a New, Combined Rapid Method Using Phage and PCR for Detection and Identification of Viable Mycobacterium paratuberculosis Bacteria within 48 Hours

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.     

The spectrum of mutations in genes associated with resistance to rifampicin,isoniazid, and fluoroquinolones in the clinical strains of <Emphasis Type="Italic">M. tuberculosis</Emphasis> reflects the transmissibility of mutant clones

To study the transmissibility of drug resistant mutant clones, M. tuberculosis samples were isolated from the patients of the clinical department and the polyclinic of the Central TB Research Institute (n = 1455) for 2011–2014. A number of clones were phenotypically resistant to rifampicin (n = 829), isoniazid (n = 968), and fluoroquinolones (n = 220). We have detected 21 resistance-associated variants in eight codons of rpoB, six variants in three codons of katG, three variants in two positions of inhA, four variants in four positions of ahpC, and nine variants in five codons of gyrA, which were represented in the analyzed samples with varied frequencies. Most common mutations were rpoB 531 Ser→Leu (77.93%), katG 315 (Ser→Thr) (94.11%), and gyrA 94 (Asp→Gly) (45.45%). We found that the mutations at position 15 of inhA (C→T) (frequency of 25.72%) are commonly associated with katG 315 (Ser→Thr). This association of two DNA variants may arise due to the double selection by coexposure of M. tuberculosis to isoniazid and ethionamide. The high transmissibility of mutated strains was observed, which may be explained by the minimal influence of the resistance determinants on strain viability. The high transmissibility of resistant variants may also explain the large populational prevalence of drug-resistant TB strains.     

Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy

We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10⁻⁸ M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10⁻¹¹ M solutions of PCR amplicons from M.tuberculosis in only 100 s.