Loop-mediated isothermal amplification is a promising candidate for the rapid detection of Mycobacterium tuberculosis. However, the high potential for carry-over contamination is the main obstacle to its routine use. Here, a closed tube LAMP was intended for the visual detection of Mtb to compare turbidimetric and two more favorable colorimetric methods using calcein and hydroxy naphthol blue (HNB). Additionally, a less studied dye (i.e., eriochrome black T (EBT)) was optimized in detail in the reaction for the first time. Mtb purified DNA and 30 clinical specimens were used to respectively determine the analytical and diagnostic sensitivities of each method. The turbidimetric method resulted in the best analytical sensitivity (100 fg DNA/reaction), diagnostic sensitivity and specificity (100%), and time-to-positivity of the test (15 min). However, this method is highly prone to subjective error in reading the results. Moreover, HNB-, calcein-, and EBT-LAMP could respectively detect 100 fg, 1 pg, and 1 pg DNA/reaction (the analytical sensitivities) in 30, 15, and 30 min, while the diagnostic sensitivity and specificity were respectively 93.3% and 100% for them all. Interestingly, EBT-LAMP showed the lowest potential for subjective error in reading the results. This report helps judiciously choose the most appropriate visual method, taking a step forward toward the field applicability of LAMP for the detection of Mtb, particularly in resource-limited settings.

Background: Pathogenic mycobacteria are a major cause of human morbidity and mortality. Mycobacterium tuberculosis is an etiological agent of human tuberculosis (TB). Designing new vaccines, including DNA vaccines, may be a useful strategy for preventing TB. Objectives: The purpose of this study was to design and construct an eukaryotic expression vector containing M. tuberculosis. Materials and Methods: Genomic DNA of M. tuberculosis H37Rv cultured on Lowenstein Jensen medium was extracted, and cfp10 was amplified by PCR. After digesting the PCR product and the plasmid, the cfp10 fragment was ligated into the vector pcDNA3.1 (+). Correct insertion was confirmed by colony PCR, restriction enzyme digestion, and sequencing. Results: Electrophoresis of the PCR product on gel showed a 303-bp target fragment. Colony PCR, restriction enzyme digestion, and Sequencing methods confirmed the accuracy of the gene cloning. Colony PCR and restriction enzyme digestion confirmed the cloning. Conclusions: Cloning of cfp10 of M. tuberculosis into an eukaryotic expression vector was performed successfully. We propose this recombinant plasmid for inducing immunity in animal models in future studies. This recombinant vector can also be used in the construction of fusion proteins. Keywords: Recombinant DNA; CFP-10 protein; Mycobacterium tuberculosis

Background: Staphylococcus aureus is a major human pathogen. Due to high prevalence of S. aureus infections and increasing resistance to antibiotics, physicians have been facing problems in choosing the appropriate empirical antibiotic therapy for such infections. Objectives: The aim of this study was to determine the pattern of antibiotic resistance in S. aureus clinical isolates of Mashhad Quaem Hospital between 2009 and 2011. Materials and Methods: In this study 170 isolates of Staphylococcus aureus were identified in laboratory. The specimens (including 82 urine, 43 wound, 37blood, 8 stool samples) were collected and examined by standard diagnostic methods. Determination of S. aureus sensitivity to antibiotics was performed using standard disc diffusion method. The antibiotic sensitivity of bacteria were reported according to the clinical laboratory standards institute (CLSI) manual as sensitive (S), intermediate (I) and resistant (R). Results: Among tested antibiotics, Staphylococci isolates were highly resistant against Ceftazidime (94%), followed by Penicillin (91%), Ampicillin (82%), Cefotaxime (65%), Erythromycin (60%), and Oxacillin (43%). Nearly all strains were susceptible to tested Vancomycin. Conclusions: Our results are similar to the reports from other parts of Iran. According to this study, resistance pattern among Staphylococcus aureus strains were widespread in Quaem hospital. The implication of this high resistance is that Staphylococcus aureus infections should be treated more consciously and not with Penicillin and other ineffective antibiotics. Keywords: Antibiotics, Antineoplastic Agents; Drug Resistance; Staphylococcus aureus