[A rapid screening program on the resistance to streptomycin and ethambutol in Mycobacterium tuberculosis isolates by PCR melting curve analysis].

OBJECTIVE: To evaluate the effects of PCR melting curve analysis assay on a rapid screening program regarding the resistance of Mycobacterium tuberculosis (MTB) clinical isolates to streptomycin and ethambutol. METHODS: A total of 331 clinical isolates of MTB had been collected since 2007-2009 in Shenzhen. Mutations at codon 306, 378-380, 406 and 497 of embB gene, codon 43, 88 of rpsL gene, and 513-517, 905-908 region of rrs gene were detected by PCR melting curve analysis. Results were compared with that of conventional drug susceptibility test. RESULTS: Compared to drug susceptibility test, sensitivity, specificity and accuracy for streptomycin resistance were 78.6%, 90.1% and 86.7%, respectively while 83.0%, 93.3% and 91.8%, respectively for ethambutol resistance detected by PCR melting curve analysis. PCR melting curve method was in good agreement with drug susceptibility test. CONCLUSION: PCR melting curve analysis on genetic regions associated with resistance to streptomycin and ethambutol seemed to be a rapid, specific and closed-tube method so it could be used for detection of streptomycin and ethambutol resistance in MTB.

[Establishment and application of a novel method for detecting MLL fusion genes of acute leukemia].

This study was aimed to establish an efficient method to detect 10 common MLL fusion genes in patients with acute leukemia. Firstly, the relevant references and databases were searched to thoroughly investigate all fusion breakpoints; the primers and probes were designed according to nearly all the involved fusion types of gene. Then the multiplex real-time PCR system was established and optimized by using the established 16 positive plasmids and negative cell lines. Finally, the detection system was clinically evaluated by means of collected 54 samples of leukemia. The results indicated that the established detection system could efficiently detect all positive plasmids with sensibility to 10 copies. Four kinds of fusion gene types such as MLL-AF4, MLL-AF9, MLL-AF10, MLL-ELL could be detected in 54 samples, the sequencing of positive samples showed consistency of sequencing results with detection results. It is concluded that a novel multiplex real-time PCR detection method is established which can detect 10 common MLL fusion genes covering about 90% of the cases harboring MLL fusions. This method is fast, sensitive, specific and reliable, and should be an useful clinical tool for identification and management of leukemia patients with MLL fusions.

[Evaluation of methods for detection of NPM1 gene mutations in acute myeloid leukemia].

The purpose of this study was to establish real-time based methods for detection of NPM1 gene mutation in acute myeloid leukemia (AML). Primers/probes were designed according to the clustered region of NPM1 mutations on exon 12. Two real-time PCR assays, including high resolution melting curve (HRM) and allele-specific PCR (AS-PCR), were developed and clinically evaluated with 89 AML samples, which were parallelly detected by capillary electrophoresis (CE) and sequencing. The results showed that a total of 17 mutation-positive samples were detected, including type A (15 cases), type B (1 case) and type Nm (1 case). HRM assay could detect all mutant types, and the analytical sensitivity was around 5%. In contrast, AS-PCR assay detected only 95% mutant types, but its sensitivity was as high as 0.01%. It is concluded that considering the characteristics of each method as well as the clinical evaluation results, HRM may be used for screening of NPM1 mutations at diagnosis, while the AS-PCR can be used for the MRD quantification during follow-up.

[Analyzing the mutations of rpoB gene in Mycobacterium tuberculosis clinical isolates by probe melting analysis assay].

OBJECTIVE: To evaluate the clinical performance of a probe melting analysis (PMA)-based real-time PCR detection kit in rapid detection of rifampin-resistant mutations in Mycobacterium tuberculosis (MTB). METHODS: The specificity of the assay was evaluated by detecting 37 non-tuberculous mycobacteria (NTM), and the detection limit of the method was evaluated by genomic DNA of a standard strain H37Rv. Finally, 962 clinical isolates were analyzed with the PMA assay by detecting mutations in rifampin resistance-determining region (RRDR) of rpoB gene, and results were verified with DNA sequencing. RESULTS: Among 37 NTM strains, three strains showed drug resistant mutation signals. The PMA method could detect down to 30 bacteria per reaction. Sample analysis showed that 186 of 962 isolates were mutants, 751 isolates were wild type and 25 isolates failed to give amplification signals. Among the mutant samples detected, 112 samples from November 2009 to April 2010 were further analyzed by sequencing, as well as 200 wild-type samples. The results showed a complete agreement with the PMA assay except for 5 samples failed in sequence analysis. CONCLUSION: The PMA assay is rapid, accurate and easy-to-use, and thus can be used for detection of rifampin-resistant in clinical isolate samples.

[Rapid detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis using real-time PCR and melting curve analysis].

OBJECTIVE: To evaluate the application of a real-time PCR and melting curve analysis assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis (M. tuberculosis). METHODS: A total of 311 clinical isolates of M. tuberculosis obtained from Shenzhen Center for Chronic Disease Control (SZCCC) were included in the study. These isolates were collected originally from national baseline survey on drug-resistant tuberculosis, project for drug resistance surveillance in Shenzhen and clinical patients in SZCCC between 2007 and 2009. rpoB gene resistance-determining region, ahpC promoter (-44 to -30 and -15 to -3), inhA promoter (-17 to -8), inhA 94 and katG 315 were detected by melting curve analysis after real-time PCR, and the results were compared with that of proportion method and DNA sequencing. The performances of the assay in detecting the resistance of rifampin and isoniazid were compared to that of reference proportion method drug susceptibility test. RESULTS: Real-time PCR and melting analysis was a closed-tube assay that could be completed within 2 - 3 h. Compared to the results of the proportion method, the sensitivity, specificity and accuracy of the assay for rifampin resistance were 97.8%, 97.1% and 97.4% respectively, and for isoniazid resistance were 86.6%, 98.7% and 92.6% respectively. CONCLUSIONS: Real-time PCR and melting analysis is a rapid, accurate and closed-tube method that can be used as a screening test for rapid identification of Multidrug-resistant tuberculosis.

[Application study on multicolor combinational probe coding real-time PCR in detection of foodborne pathogens].

OBJECTIVE: To investigate the detection limit of multicolor combinational probe coding real-time PCR (MCPC-PCR) in detection of Salmonella and Staphylococcus aureus suspended in the food samples, and to apply MCPC-PCR to detect the samples of food poisoning. METHODS: Series concentration of bacterium suspension (10(1) - 10(9) CFU/ml) was prepared by using 22 simulated samples including fresh meat and cakes and then MCPC-PCR was applied to detect Salmonella and Staphylococcus aureus in 22 samples. Enrichment broth of 101 frozen samples and 5 early patients' anal swabs in food poisoning cases were detected after the DNA samples were extracted. RESULTS: The limits of MCPC-PCR assay in detecting Salmonella and Staphylococcus aureus were about 10(2) copies/test; 101 frozen enrichment broth of samples in food poisoning cases were detected by MCPC-PCR assay, of 23 positive samples, 18 were confirmed by bacteriology techniques; 96 samples detected by MCPC-PCR and bacteriology techniques had the same results, and the coincidence rate was 95.05%. Anal swabs, collected from 5 of early patients in a food poisoning case gave a clue to be Vibrio parahaemolyticus by MCPC-PCR assay and then were perfectly consistent with bacteriology assay. CONCLUSION: As a method of high sensitivity and good specificity, MCPC-PCR assay can quickly and conveniently detect multiple pathogens existing in food samples, therefore we recommend it to be used in rapidly screening or simultaneous detection of food-borne diseases.

Quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea, using a molecular beacon.

A real-time polymerase chain reaction (PCR) assay utilizing a molecular beacon for the quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea (LYCIV), was developed, which involved the amplification of a 122bp DNA fragment from a conserved region of LYCIV ATPase gene. The specific probe consisting of two short arm and a central loop sequences complementary to the target amplicon was characterized with respect to its efficiency of quenching (E(ff)), and signal to background ratio by spectrofluorometric analysis of its hybridization with the complementary oligonucleotide target. The positive control plasmid pFHT-ATPase containing the target sequence was quantified to make the standard curve for sample detection after serial 10-fold dilution. Linear coefficient correlations between cycle threshold (C(T)) value and logarithmic positive plasmid concentration were close to one (r(2)=0.998) and the detection limit of the assay was 70 copies of positive plasmid/assay. The specificity of this real-time PCR was also demonstrated by using the genomic DNA templates from the healthy fish, white spot syndrome baculovirus (WSSV), and epizootic heamatopietic necrosis virus (EHNV), respectively. The coefficient of variation (CV) of the assay ranged from 1.16 to 4.42%, depending on the concentration of the positive plasmid. The quantitative detection of different tissues from LYCIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (6.86 x 10(6) and 4.62 x 10(6) viral genome copies/mg tissue, respectively) while no viral DNA was detected in the muscular tissue. These results suggested that the real-time PCR assay reported here could be used for rapid, sensitive, and quantitative detection of LYCIV infection.

[Rapid simultaneous detection of Salmonella and Shigella using modified molecular beacons and real-time PCR].

OBJECTIVE: Dual detection of Salmonella and Shigella using modified molecular beacons and real-time PCR was developed. The established method was applied to rapid diagnosis of Salmonella and Shigella' food poisoning, and for routine monitoring programs. METHODS: Two sets of primers were designed based on the core sequence of invA gene and ssaR gene published on GenBank to detect Salmonella, and ipaH gene were selected to detect Shigella. Three corresponding modified molecular beacons labeled with different fluorophors were designed. The molecular beacons and primer sets were tested against numerous strains from 55 different bacterial species. Then the two assays were combined to establish the dual real-time PCR assay, and were applied to the food poisoning diagnosis and surveillance. RESULTS: For the modified molecular beacons-based dual real-time PCR assay, the sensitivity achieved was 69-93 fg/microl, 32-64 CFU/ml or 1-2 CFU/PCR reaction. There was no cross-reaction with other bacteria served as control. The dual real-time PCR assay was used to detect 134 Salmonella strains and 67 Shigella strains but no false signals were observed. 1100 food poisoning samples were tested with 569 Salmonella and 42 were Shigella identified by real time PCR. Among the positive samples, 551 were detected Salmonella and 41 were Shigella by traditional culture method. The overall test could be finished within 2 hours to one day starting from sample preparation. CONCLUSION: The modified molecular beacons-based dual real-time PCR assay was rapid, sensitive, and specific. It could be applied to the rapid diagnosis of Salmonella and Shigella' food poisoning.

[Construction of RNA-containing virus-like nanoparticles expression vector with cysteine residues on surface and fluorescent decoration].

Site-directed mutagenesis was performed at the codon 15 of the MS2 bacteriophage coat protein gene,which had been cloned to the virus-like particles expression vector containing non-self RNA fragment. The produced expression vector,termed pARSC, was transformed to E. coli DH5alpha. The positive clones were selected and proliferated. The harvested cells were treated with sonication and the supernatant was then subjected to linear sucrose density gradients centrifugation (15% to 60%) at 32000 r/min for 4 h at 4 degrees C. The virus-like particles, VLP-Cy, were collected at 35% sucrose density. The particles were examined by transmission electron microscopy and the spherical viral particles of approximately 27 nm in diameter were found. The thiolated VLP-Cy was then chemically modified with fluorescein -5'-maleimide. The covalent fluorescent labeling was confirmed by absorption analysis, SDS-PAGE and MALDI-TOF mass spectroscopy. This is the first report of preparation of RNA-containing natural fluorescent nanoparticles. The study highlight the versatility of MS2 bacteriophage capsids as building blocks for functional nanomaterials construction for a variety of application purposes.

[Detection of APC mutation by real-time polymerase chain reaction and melting curve analysis].

OBJECTIVE: To establish a simple, reliable and low cost approach for clinical detection of APC mutation. METHODS: Using SYBR Green I as the real-time polymerase chain reaction (PCR) product indicator, a DNA fragment of 270 bp targeting APC_1309 mutation (5 bp deletion) was amplified from the sample DNA. A short fragment (40/35 bp) was then amplified from the 270 bp PCR product, followed by melting curve analysis from 65 degrees C to 99 degrees C at 0.5 degrees C/step. RESULTS: A total of 18 paraffin-embedded tumor samples were analyzed, of which 7 were tested positive for the mutation and 11 were negative. No mutation was detected in any of the 20 normal peripheral blood samples. CONCLUSIONS: Real-time PCR melting curve analysis can be used for routine APC mutation detection. The simple design, low cost and high reliability should allow similar applications to the analysis of a variety of other gene mutations.

[Screening C282Y mutation with double-stranded probes using synchronous fluorometry].

We described in the paper a new high-throughput screening method for Cys282Tyr mutation in hereditary haemochromatosis with double-stranded probe using synchronous fluorometry. The probe for wild type was labeled with Fam, the probe for mutant type was labeled with Joe. After PCR, reaction tubes were transferred to a spectrofluorometer, where synchronous spectra were scanned in a constant-wavelength mode. The genotype could be obtained through the appearance of the fluorescence peaks corresponding to each probe. The results were totally in agreement with restriction endonuclease analysis. Considering the simplicity,low cost and specificity, this approach could be generally applied to detect varieties of gene mutations.