The Recent Transmission and Associated Risk Factor of Mycobacterium tuberculosis in Golmud City, China.

Background: Tuberculosis (TB) remains a severe public health problem globally, and it is essential to comprehend the transmission pattern to control tuberculosis. Herein, we evaluated the drug-resistant characteristics, recent transmission, and associated risk factors of TB in Golmud, Qinghai, China. Methods: In this study, we performed a population-based study of patients diagnosed with TB in Golmud from 2013 to 2018. Drug-susceptibility testing and whole-genome sequencing were performed on 133 Mycobacterium tuberculosis strains. The genomic clustering rate was calculated to evaluate the level of recent transmission. Risk factors were identified by logistic regression analysis. Results: Our results showed that 46.97% (62/132) of strains were phylogenetically clustered and formed into 23 transmission clusters, suggesting a high recent transmission of TB in the area. 12.78% (17/133) strains were multidrug-resistant/rifampicin tuberculosis (MDR/RR-TB), with a high drug-resistant burden. Based on drug resistance gene analysis, we found 23 strains belonging to genotype MDR/RR-TB, where some strains may have borderline mutations. Among these strains, 65.2% (15/23) were found within putative transmission clusters. Additionally, risk factor analysis showed that recent transmission of TB happened more in patients with Tibetan nationality or older age. Conclusion: Overall our study indicates that the recent transmissions of MTB strains, especially genotypic MDR/RR strains, drive the tuberculosis epidemic in Golmud, which could contribute to developing effective TB prevention and control strategies.

Association of lineage 4.2.2 of Mycobacterium tuberculosis with the 63-bp deletion variant of the mpt64 gene.

IMPORTANCE: To date, rapid diagnostic methods based on the MPT64 antigen assay are increasingly utilized to differentiate between non-tuberculous mycobacteria and TB disease in clinical settings. Furthermore, numerous novel techniques based on the MPT64 release assay are continuously being developed and applied for the identification of both pulmonary and extrapulmonary TB. However, the diagnostic accuracy of the MPT64 antigen assay is influenced by the presence of 63 bp deletion variants within the mpt64 gene. To our knowledge, this is the first report on the association between the 63 bp deletion variant in mpt64 and Mycobacterium tuberculosis L4.2.2 globally, which highlights the need for the cautious utilization of MPT64-based testing in regions where L4.2.2 isolates are prevalent, such as China and Vietnam, and MPT64 negative results should be confirmed with another assay. In addition, further studies on vaccine development and immunology based on MPT64 should consider these isolates with 63 bp deletion variant.

Mn3O4/CuOx heterostructure for nitrate electroreduction to ammonia.

Here, we designed a Mn3O4/CuOx heterostructure supported on copper foil (CF) for electrocatalytic nitrate reduction to ammonia. The selectivity and Faraday efficiency of ammonia were 96.79% and 86.55%, respectively. Multiple characterizations revealed that Mn3O4/CuOx/CF showed faster charge transfer and created more electron-deficient Mn sites, electron-rich Cu sites and large numbers of oxygen vacancies, which were conducive to improving the catalytic activity. This work may open an avenue for the construction of heterostructures as an electrocatalyst for the reduction of nitrate to ammonia.

Endogenous relapse and exogenous reinfection in recurrent pulmonary tuberculosis: A retrospective study revealed by whole genome sequencing.

Background: Tuberculosis may reoccur due to reinfection or relapse after initially successful treatment. Distinguishing the cause of TB recurrence is crucial to guide TB control and treatment. This study aimed to investigate the source of TB recurrence and risk factors related to relapse in Hunan province, a high TB burden region in southern China. Methods: A population-based retrospective study was conducted on all culture-positive TB cases in Hunan province, China from 2013 to 2020. Phenotypic drug susceptibility testing and whole-genome sequencing were used to detect drug resistance and distinguish between relapse and reinfection. Pearson chi-square test and Fisher exact test were applied to compare differences in categorical variables between relapse and reinfection. The Kaplan-Meier curve was generated in R studio (4.0.4) to describe and compare the time to recurrence between different groups. p < 0.05 was considered statistically significant. Results: Of 36 recurrent events, 27 (75.0%, 27/36) paired isolates were caused by relapse, and reinfection accounted for 25.0% (9/36) of recurrent cases. No significant difference in characteristics was observed between relapse and reinfection (all p > 0.05). In addition, TB relapse occurs earlier in patients of Tu ethnicity compared to patients of Han ethnicity (p < 0.0001), whereas no significant differences in the time interval to relapse were noted in other groups. Moreover, 83.3% (30/36) of TB recurrence occurred within 3 years. Overall, these recurrent TB isolates were predominantly pan-susceptible strains (71.0%, 49/69), followed by DR-TB (17.4%, 12/69) and MDR-TB (11.6%, 8/69), with mutations mainly in codon 450 of the rpoB gene and codon 315 of the katG gene. 11.1% (3/27) of relapse cases had acquired new resistance during treatment, with fluoroquinolone resistance occurring most frequently (7.4%, 2/27), both with mutations in codon 94 of gyrA. Conclusion: Endogenous relapse is the main mechanism leading to TB recurrences in Hunan province. Given that TB recurrences can occur more than 4 years after treatment completion, it is necessary to extend the post-treatment follow-up period to achieve better management of TB patients. Moreover, the relatively high frequency of fluoroquinolone resistance in the second episode of relapse suggests that fluoroquinolones should be used with caution when treating TB cases with relapse, preferably guided by DST results.

High diversity of clinical Mycobacterium intracellulare in China revealed by whole genome sequencing.

Mycobacterium intracellulare is the most common cause of nontuberculous mycobacterial lung disease, with a rapidly growing prevalence worldwide. In this study, we performed comparative genomic analysis and antimicrobial susceptibility characteristics analysis of 117 clinical M. intracellulare strains in China. Phylogenetic analysis showed that clinical M. intracellulare strains had high genetic diversity and were not related to the geographical area. Notably, most strains (76.07%, 89/117) belonged to Mycobacterium paraintracellulare (MP) and Mycobacterium indicus pranii (MIP) in the genome, and we named them MP-MIP strains. These MP-MIP strains may be regarded as a causative agent of chronic lung disease. Furthermore, our data demonstrated that clarithromycin, amikacin, and rifabutin showed strong antimicrobial activity against both M. intracellulare and MP-MIP strains in vitro. Our findings also showed that there was no clear correlation between the rrs, rrl, and DNA gyrase genes (gyrA and gyrB) and the aminoglycosides, macrolides, and moxifloxacin resistance, respectively. In conclusion, this study highlights the high diversity of M. intracellulare in the clinical setting and suggests paying great attention to the lung disease caused by MP-MIP.

Transmission and Drug Resistance Genotype of Multidrug-Resistant or Rifampicin-Resistant Mycobacterium tuberculosis in Chongqing, China.

Multidrug-resistant or rifampicin-resistant tuberculosis (MDR/RR-TB) is a global barrier for the Stop TB plan. To identify risk factors for treatment outcome and cluster transmission of MDR/RR-TB, whole-genome sequencing (WGS) data of isolates from patients of the Chongqing Tuberculosis Control Institute were used for phylogenetic classifications, resistance predictions, and cluster analysis. A total of 223 MDR/RR-TB cases were recorded between 1 January 2018 and 31 December 2020. Elderly patients and those with lung cavitation are at increased risk of death due to MDR/RR-TB. A total of 187 MDR/RR strains were obtained from WGS data; 152 were classified as lineage 2 strains. Eighty (42.8%) strains differing by a distance of 12 or fewer single nucleotide polymorphisms were classified as 20 genomic clusters, indicating recent transmission. Patients infected with lineage 2 strains or those with occupations listed as "other" are significantly associated with a transmission cluster of MDR/RR-TB. Analysis of resistant mutations against first-line tuberculosis drugs found that 76 (95.0%) of all 80 strains had the same mutations within each cluster. A total of 55.0% (44 of 80) of the MDR/RR-TB strains accumulated additional drug resistance mutations along the transmission chain, especially against fluoroquinolones (63.6% [28 of 44]). Recent transmission of MDR/RR strains is driving the MDR/RR-TB epidemics, leading to the accumulation of more serious resistance along the transmission chains. IMPORTANCE The drug resistance molecular characteristics of MDR/RR-TB were elucidated by genome-wide analysis, and risk factors for death by MDR/RR-TB were identified in combination with patient information. Cluster characteristics of MDR/RR-TB in the region were analyzed by genome-wide analysis, and risk factors for cluster transmission (recent transmission) were analyzed. These analyses provide reference for the prevention and treatment of MDR/RR-TB in Chongqing.

Identification and drug susceptibility testing of the subspecies of Mycobacterium avium complex clinical isolates in mainland China.

OBJECTIVES: The Mycobacterium avium complex (MAC), comprising a series of subspecies, has a worldwide distribution, with differences in drug susceptibility among subspecies. This study aimed to assess the composition of MAC and susceptibility differences among subspecies in mainland China. METHODS: A total of 287 MAC clinical strains were included in the study. Multitarget sequences were applied to accurately identify subspecies, and a microdilution method was used to evaluate minimum inhibitory concentrations (MICs) among subspecies using Sensititre SLOMYCO plates. RESULTS: Mycobacterium intracellular (N = 169), Mycobacterium avium (N = 52), Mycobacterium chimaera (N = 22), Mycobacterium marseillense (N = 25), Mycobacterium colombiense (N = 14), Mycobacterium yongonense (N = 4), Mycobacterium vulneris (N = 3) and Mycobacterium timonense (N = 2) were isolated from MAC. Clarithromycin, amikacin and rifabutin showed lower MIC50 and MIC90 values than other drugs, and the resistance rates of clarithromycin, amikacin, linezolid and moxifloxacin were 6.3%, 10.5%, 51.9% and 46.3%, respectively. The resistance rates of clarithromycin and moxifloxacin in the initial treatment group were significantly lower than those in the retreatment group (4.09% vs. 12.94%; 30.41% vs. 75.29%; P < 0.05). Drug susceptibility differences were observed in clarithromycin and moxifloxacin among the five major subspecies (P < 0.05); however, those statistically significant differences disappeared when MACs were divided into two groups according to previous anti-tuberculosis (anti-TB) treatment history. CONCLUSION: This study revealed that MAC, primarily comprising M. intracellulare, was susceptible to clarithromycin, amikacin and rifabutin. Drug susceptibility among subspecies did not exhibit intrinsic differences in our study. Previous anti-TB treatment patients are more resistant to drugs; thus, attention should be given to those patients in the clinic.

Drug-Resistant Characteristics, Genetic Diversity, and Transmission Dynamics of Rifampicin-Resistant Mycobacterium tuberculosis in Hunan, China, Revealed by Whole-Genome Sequencing.

To gain a deep insight into the additional drug-resistant profiles, genetic diversity, and transmission dynamics of rifampicin-resistant tuberculosis (RR-TB) circulating in Hunan province, drug susceptibility testing and whole-genome-sequencing were performed among RR-TB strains collected from Jan. 2013 to Jun. 2018 in Hunan province. A total of 124 RR-TB strains were recovered successfully and included into the final analysis. Lineage 2.2.1 was the dominant sublineage, accounting for 72.6% (90/124), followed by lineage 4.5 (11.3%, 14/124), lineage 4.4 (8.1%, 10/124), lineage 4.2 (6.5%, 8/124) and lineage 2.2.2 (1.6%, 2/124). Overall, 83.1% (103/124) and 3.2% (4/124) of RR-TB were MDR-TB and XDR-TB, respectively. Nearly 30% of RR-TB isolates were resistant to fluoroquinolones, and 26.6% (33/124) were pre-XDR-TB. Moreover, 30.6% (38/124) of RR-TB strains were identified as phenotypically resistance to pyrazinamide. Totally, 17 clusters containing 48 (38.7%, 48/124) RR-TB strains were identified, ranging in size from 2 to 10 isolates. No significant difference was detected in clustering rate between lineage 2 and lineage 4 (χ2 = 0.027, P = 0.870). Our study revealed the complexity of RR-TB strains circulating in Hunan province with complex additional drug-resistant profile and relatively higher clustering rates. Comprehensive information based on WGS should be used to guide the design of treatment regimens and tailor public interventions. IMPORTANCE Comprehensive information such as genetic background and drug-resistant profile of MTB strains could help to tailor public interventions. However, these data are limited in Hunan province, one of the provinces with high-TB burden in China. So, this study aimed to provide us with deep insight into the molecular epidemiology of RR-TB isolates circulating in Hunan province by combining phenotypic drug susceptibility testing and whole-genome sequencing. To our knowledge, this is the first study to use whole-genome sequencing data of RR-TB strains spanning more than 5 years for molecular epidemiology analysis in Hunan province, which allows us to identify genetic background information and clustered strains more accurately. Our study revealed the complexity of RR-TB strains circulating in Hunan province with complex additional drug-resistant profile and relatively higher clustering rates. Comprehensive information based on WGS should be used to guide the design of treatment regimens and tailor public interventions.

Prevalence of Mycobacterium tuberculosis resistant to bedaquiline and delamanid in China.

OBJECTIVES: The new antituberculous drugs delamanid and bedaquiline form the last line of defence against drug-resistant tuberculosis (TB). Understanding the background prevalence of resistance to new drugs can help predict the lifetime of these drugs' effectiveness and inform regimen design. METHODS: Mycobacterium tuberculosis without prior exposure to novel anti-TB drugs were analysed retrospectively. Drug susceptibility testing for bedaquiline, delamanid, linezolid, clofazimine and widely used first- and second-line anti-TB drugs was performed. All TB isolates with resistance to new or repurposed drugs were subjected to whole-genome sequencing to explore the molecular characteristics of resistance and to perform phylogenetic analysis. RESULTS: Overall, resistance to delamanid, bedaquiline, linezolid and clofazimine was observed in 0.7% (11/1603), 0.4% (6/1603), 0.4% (7/1603) and 0.4% (6/1603) of TB isolates, respectively. Moreover, 1.0% (1/102), 2.9% (3/102), 3.9% (4/102) and 1.0% (1/102) of multidrug-resistant TB (MDR-TB) were resistant to bedaquiline, delamanid, linezolid and clofazimine, respectively. Whereas 22.2% (2/9) of extensively-drug resistant tuberculosis (XDR-TB) isolates were resistant to both delamanid and linezolid, and none was resistant to bedaquiline or clofazimine. Phylogenetic analysis showed that recent transmission occurred in two XDR-TB with additional resistance to delamanid and linezolid. None known gene mutation associated with delamanid resistance was detected. All four isolates with cross-resistance to bedaquiline and clofazimine had a detected gene mutation in Rv0678. Three of five strains with linezolid resistance had a detected gene mutation in rplC. CONCLUSION: Detection of resistance to new anti-TB drugs emphasises the pressing need for intensive surveillance for such resistance before their wide usage.

Geniposide alleviates lipopolysaccharide-caused apoptosis of murine kidney podocytes by activating Ras/Raf/MEK/ERK-mediated cell autophagy.

Proteinuria is one of the most important clinical features of nephrotic syndrome (NS). Injury of podocyte has been proved to contribute to the occurrence of proteinuria. This study explored the effects of geniposide (GEN) on lipopolysaccharide (LPS)-caused murine kidney podocyte MPC5 apoptosis and autophagy. Viability and apoptosis of MPC5 cells were respectively detected with the help of CCK-8 assay and Guava Nexin assay. 3-Methyladenine (3-MA) was used as an autophagy inhibitor, while rapamycin as autophagy activator. Si-Beclin-1 was transfected in MPC5 cells to down-regulate the expression of Beclin-1. We found that LPS stimulation significantly caused MPC5 cell viability reduction, apoptosis and autophagy (P < .05 or P < .01). GEN treatment remarkably alleviated the LPS-caused MPC5 cell viability reduction and apoptosis, but promoted cell autophagy (P < .05). Moreover, 3-MA incubation or si-Beclin-1 transfection notably weakened the effects of GEN on LPS-caused MPC5 cell apoptosis and autophagy (P < .05), while rapamycin had opposite effects (P < .05). Furthermore, GEN activated Ras/Raf/MEK/ERK pathway in LPS-treated MPC5 cells (P < .05). In conclusion, this research verified the protective effects of GEN on podocytes damage. GEN alleviates LPS-caused apoptosis of murine kidney podocytes by activating Ras/Raf/MEK/ERK-mediated cell autophagy. Highlights: LPS causes podocyte MPC5 apoptosis and autophagy. GEN alleviates LPS-caused MPC5 cell apoptosis, but promotes cell autophagy. 3-MA or si-Beclin-1 weakens the effects of GEN on LPS-treated MPC5 cells. Rapamycin strengthens the effects of GEN on LPS-treated MPC5 cells. GEN activates Ras/Raf/MEK/ERK pathway in LPS-treated MPC5 cells.

China's tuberculosis epidemic stems from historical expansion of four strains of Mycobacterium tuberculosis.

A small number of high-burden countries account for the majority of tuberculosis cases worldwide. Detailed data are lacking from these regions. To explore the evolutionary history of Mycobacterium tuberculosis in China-the country with the third highest tuberculosis burden-we analysed a countrywide collection of 4,578 isolates. Little genetic diversity was detected, with 99.4% of the bacterial population belonging to lineage 2 and three sublineages of lineage 4. The deeply rooted phylogenetic positions and geographic restriction of these four genotypes indicate that their populations expanded in situ following a small number of introductions to China. Coalescent analyses suggest that these bacterial subpopulations emerged in China around 1,000 years ago, and expanded in parallel from the twelfth century onwards, and that the whole population peaked in the late eighteenth century. More recently, sublineage L2.3, which is indigenous to China and exhibited relatively high transmissibility and extensive global dissemination, came to dominate the population dynamics of M. tuberculosis in China. Our results indicate that historical expansion of four M. tuberculosis strains shaped the current tuberculosis epidemic in China, and highlight the long-term genetic continuity of the indigenous M. tuberculosis population.

A novel isothermal amplification-based method to detect Mycobacterium tuberculosis complex.

Mycobacterium tuberculosis (MTB) is the causative agent of pulmonary tuberculosis. Rapid and accurate diagnosis is crucial to tuberculosis control and prevention. A series of diagnostic methods has been available for MTB detection; however, new rapid, simple and affordable methods are needed. In this study, a multiple cross displacement amplification (MCDA)-based assay was developed to detect the IS6110 gene of the M. tuberculosis complex. Hydroxy naphthol blue (HNB), a colorimetric indicator, was used to detect amplification products. Amplification was carried out at a constant temperature (68°C) for only 40min, followed by direct determination of amplification products through observation of color variations. The entire detection procedure, from processing of specimens to reading of results, required only 85min. Moreover, this assay, hereafter designated MTB-MCDA-HNB, was able to detect as little as 1pg of DNA extracted from the Bacille Calmette-Guerin (BCG) strain of Mycobacterium bovis. No cross-reaction with nontuberculous mycobacteria (NTM) species was observed. Moreover, during testing of clinical samples, the sensitivity and specificity of MCDA results were 94.7% and 92.9%, respectively, when compared to results obtained using the Xpert MTB/RIF method. Therefore, the MTB-MCDA-HNB method developed in this study holds promise for application as an effective point-of-care test to detect M. tuberculosis.

Controlled synthesis of hierarchically crossed metal oxide nanosheet arrays for diesel soot elimination.

We propose a facile and versatile strategy for the fabrication of a hierarchically crossed metal oxide nanosheet array on a monolithic FeOx substrate. This strategy can also be extended to synthesize a composite metal oxide nanosheet array, in which the composition can be easily modulated. The as-obtained hierarchically crossed metal oxide nanosheet array exhibited high catalytic activities for diesel soot elimination owing to the enhanced contact efficiency between soot particulates and macroporous catalysts.

Ligand Selectivity Mechanism and Conformational Changes in Guanine Riboswitch by Molecular Dynamics Simulations and Free Energy Calculations.

Riboswitches regulate gene expression through direct and specific interactions with small metabolite molecules. Binding of a ligand to its RNA target is high selectivity and affinity and induces conformational changes of the RNA's secondary and tertiary structure. The structural difference of two purine riboswitches aptamers is caused by only one single mutation, where cytosine 74 in the guanine riboswitch is corresponding to a uracil 74 in adenine riboswitch. Here we employed molecular dynamics (MD) simulation, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and thermodynamic integration computational methodologies to evaluate the energetic and conformational changes of ligands binding to purine riboswitches. The snapshots used in MM-PBSA calculation were extracted from ten 50 ns MD simulation trajectories for each complex. These free energy results are in consistent with the experimental data and rationalize the selectivity of the riboswitches for different ligands. In particular, it is found that the loss in binding free energy upon mutation is mainly electrostatic in guanine (GUA) and riboswitch complex. Furthermore, new hydrogen bonds are found in mutated complexes. To reveal the conformational properties of guanine riboswitch, we performed a total of 6 µs MD simulations in both the presence and the absence of the ligand GUA. The MD simulations suggest that the conformation of guanine riboswitch depends on the distance of two groups in the binding pocket of ligand. The conformation is in a close conformation when U51-A52 is close to C74-U75.

Computational Studies of a Mechanism for Binding and Drug Resistance in the Wild Type and Four Mutations of HIV-1 Protease with a GRL-0519 Inhibitor.

Drug resistance of mutations in HIV-1 protease (PR) is the most severe challenge to the long-term efficacy of HIV-1 PR inhibitor in highly active antiretroviral therapy. To elucidate the molecular mechanism of drug resistance associated with mutations (D30N, I50V, I54M, and V82A) and inhibitor (GRL-0519) complexes, we have performed five molecular dynamics (MD) simulations and calculated the binding free energies using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. The ranking of calculated binding free energies is in accordance with the experimental data. The free energy spectra of each residue and inhibitor interaction for all complexes show a similar binding model. Analysis based on the MD trajectories and contribution of each residues show that groups R2 and R3 mainly contribute van der Waals energies, while groups R1 and R4 contribute electrostatic interaction by hydrogen bonds. The drug resistance of D30N can be attributed to the decline in binding affinity of residues 28 and 29. The size of Val50 is smaller than Ile50 causes the residue to move, especially in chain A. The stable hydrophobic core, including the side chain of Ile54 in the wild type (WT) complex, became unstable in I54M because the side chain of Met54 is flexible with two alternative conformations. The binding affinity of Ala82 in V82A decreases relative to Val82 in WT. The present study could provide important guidance for the design of a potent new drug resisting the mutation inhibitors.

Factors Associated with Missed Detection of Mycobacterium tuberculosis by Automated BACTEC MGIT 960 System.

Despite the demonstration of excellent performance, mycobacterial growth in BACTEC MGIT 960 can go undetected. The aim of this study was to investigate the prevalence of "false-negative" culture sample in Beijing and the potential factors associated with the detection failures by MGIT 960. Of the 577 sputum samples tested, 141 (24.4%) were culture-positive for mycobacteria, of which 133 (94.3%) were automatically determined by MGIT 960 system and 8 (5.7%) were positive for visual growth (false negative by MGIT). Statistical analysis showed that positive grade of specimen had no influence on the false-negative rate by MGIT 960 system (χ2 = 2.207, P = 0.820). In addition, the mean time to detection (TTD) was 241.4 (range: 224-261) hours for false-negative group and 186.8 (range: 173-199) hours for positive group. The difference in TTD between false-negative and positive groups was statistically significant (P < 0.01). In conclusion, our data demonstrate that the automatic MGIT missed a small portion of bacteriological mycobacterial patients. In addition, the poor growth rate rather than the low grade of AFB smear is associated with the detection failure by MGIT. Our findings highlight the notion that manual inspection for all instrument-negative MGIT tubes will bring about considerable benefit to patients and clinicians.

The Novel Multiple Inner Primers-Loop-Mediated Isothermal Amplification (MIP-LAMP) for Rapid Detection and Differentiation of Listeria monocytogenes.

Here, a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MIP-LAMP), was devised and successfully applied to detect Listeria monocytogenes. A set of 10 specific MIP-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene. The MIP-LAMP assay efficiently amplified the target element within 35 min at 63 °C and was evaluated for sensitivity and specificity. The templates were specially amplified in the presence of the genomic DNA from L. monocytogenes. The limit of detection (LoD) of MIP-LAMP assay was 62.5 fg/reaction using purified L. monocytogenes DNA. The LoD for DNA isolated from serial dilutions of L. monocytogenes cells in buffer and in milk corresponded to 2.4 CFU and 24 CFU, respectively. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by adding Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel electrophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MIP-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MIP-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains.

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification.

We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61-65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification: A Novel Analytically Rapid, Sensitive, Multiplex Loop-Mediated Isothermal Amplification Detection Technique.

Loop-mediated isothermal amplification (LAMP) is restricted to detecting a single target, limiting the usefulness of this method. To achieve multiplex LAMP-based detection, we developed a novel approach we called the multiple endonuclease restriction real-time-LAMP assay. In this system, the LAMP forward or backward inner primers contain 5' end short sequences that are recognized by the restriction endonuclease Nb.BsrDI, and the new forward or backward inner primers were modified at the 5' end with a fluorophore and in the middle with a dark quencher. Nb.BsrDI digests the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which releases the quenching, resulting in a gain of signal. The assay permitted real-time detection of single or multiple target sequences in a single tube, and the positive results can be obtained in as short as 12 minutes. The novel methodology is highly efficient and specific, detecting down to 250 fg of DNA per reaction of Listeria DNA tested, and was successful in evaluating raw meat samples. The multiple endonuclease restriction real-time-LAMP technology, which is an extension of LAMP to accommodate robust, target-specific, and multiplex detection, provides a molecular diagnostic tool with less detection time and high sensitivity and specificity compared with those of LAMP and quantitative real-time PCR.

The significant impact of polydopamine on the catalytic performance of the carried Au nanoparticles.

Mussel-inspired polydopamine catalyst carriers dramatically enhance the catalytic performance (∼450%) of Au nanoparticles in methylene blue reduction, which is attributed to the local enrichment mechanism caused by the favourable attractive interaction between the polydopamine and reactants.