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PubMed2001

Use of cryopreserved pronuclear embryos for the production of transgenic mice.

A series of experiments was conducted to test the hypothesis that an improved cryopreservation protocol for pronuclear stage mouse embryos will produce transgenic (Tg) mice by pronuclear gene injection at a rate not significantly different from noncryopreserved embryos. In the first experiment, three cryoprotective agents (CPAs) (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG]) and two cryopreservation protocols, currently used for pronuclear embryos, were compared in regard to their ability to maintain post-thaw morphological integrity and in vitro developmental competence. In the second and third experiments, the optimal cryopreservation protocol determined from the first experiment was used to evaluate in vitro developmental competence of pronuclear embryos following green fluorescence protein gene injection and in vivo developmental competence as well as the gene integration rates. Survival (morphological integrity and development to two cells) of embryos cryopreserved in the presence of DMSO was higher (P < 0.05) than those cryopreserved with either PG or EG. Postinjection developmental competence (development to two cells) of cryopreserved CBA, C57B6/JxCBA-F1 and noncryopreserved (control) embryos was not different (P > 0.05). Postinjection blastocyst formation rate of cryopreserved and noncryopreserved C57B6/JxCBA-F1 embryos was similar (P > 0.05); however, noncryopreserved CBA embryos resulted in a higher blastocyst formation than controls (P < 0.05). While there was no difference in the percentage of transgenic fetuses between cryopreserved and control CBA embryos (P > 0.05), cryopreserved C57B6/JxCBA-F1 embryos resulted in lower transgenic fetuses than control (P < 0.05). These results indicate that the use of cryopreserved mouse pronuclear embryos can be a useful and efficient approach to the production of Tg mice.

Animals↗

PubMed2026

Optimization of protoplast based DNA isolation and genome analysis in a gamma-irradiated Aspergillus niger mutant strain.

Aspergillus niger is an important industrial fungus widely used for citric acid production and a range of biotechnological applications. In this study, a protoplast-based DNA isolation protocol was optimized for a gamma-irradiated A. niger AN-L103_M1 mutant strain, followed by whole-genome sequencing and functional genome analysis. Protoplast yield was strongly influenced by enzyme concentration and the molarity of the osmotic stabilizer. The highest yield was achieved at an enzyme concentration of 50 mg/mL (2.487 ± 0.04 × 10⁸ cells/mL) and 0.8 M KCl (2.550 ± 0.06 × 10⁸ cells/mL), with both factors showing significant effects (p < 0.0001) in GraphPad Prism 11.0.0. Whole-genome sequencing performed using an Illumina NovaSeq 6000 platform yielded a 37.06 Mb draft genome assembled into 537 contigs, with an N50 of 363,084 bp and a GC content of 48.2%. BUSCO 14 analysis showed high completeness (97.95% complete BUSCOs). Functional annotation and KEGG pathway mapping identified genes involved in glycolysis, the tricarboxylic acid cycle, and citrate biosynthesis, while biosynthetic gene cluster analysis revealed diverse potential for secondary metabolite production. These findings provide an optimized workflow for protoplast-based DNA isolation and genome-scale functional analysis in A. niger, proposing a basis for future comparative genomics, transformation studies, and experimentally validated metabolic engineering.

Aspergillus niger↗

PubMed1991

Ability, disability, and the functional capacity of patients with cardiovascular disease.

Assessment of functional capacity, of ability and disability among patients with cardiovascular disease raises a number of problems and issues for which there are currently only imperfect or incomplete answers. Emphasis must be placed on the lack of predictable relationship of anatomic abnormality and functional abnormality. For example, the percentage obstruction of the coronary artery documents the anatomic extent of the disease, rather than the limitation of functional capacity; the same lack of predictive value characterizes the decrease in resting ventricular ejection fraction. The response to a challenge of activity or exertion currently appears to offer the optimal method of assessing functional capacity for work, although a brief continuous exercise test may not be the optimal exercise protocol by which to evaluate endurance. As an example, in our laboratory, comparing a low-level continuous exercise test protocol with one with an intermittent exercise design (i.e., periods of exercise alternating with periods at rest), patients typically can perform at least one additional stage of exercise on the discontinuous or intermittent test protocol. This occurred without significant differences in the final heart rate, blood pressure, or rate-pressure product, probably because most patients so tested were limited not by myocardial ischemia but by musculoskeletal problems, fatigue, or dyspnea (8). An unmet need is a comparison of exercise test protocols for the assessment of functional capacity, possibly the development of new test protocols for patients with limited functional capacity, and the evaluation of the relationship of these test data to eight hours of occupational activity in the workplace setting. It appears logical that a diagnostic exercise test should differ from one designed to determine functional capacity, but the results of a variety of exercise test protocols should be compared with the actual physical activity able to be performed in the workplace, as well as with reported symptoms. It should be defined whether testing is to be performed on optimal medical therapy, which I believe should be the case; or whether the technique used for diagnostic exercise testing, that of the minimal medication possible, is to be employed. Next, the time after surgical intervention or following a prolonged hospitalization at which to test should be delineated in that the deconditioning effect of immobilization may substantially decrease effort tolerance, unrelated to the severity of the underlying cardiovascular disease. Finally, should exercise rehabilitation be recommended or required before testing for cardiovascular impairment; major improvement in functional capacity has occurred in previously sedentary patients with a variety of cardiovascular diseases, including those with important manifestations of myocardial ischemia and ventricular dysfunction.(ABSTRACT TRUNCATED AT 400 WORDS)

Cardiovascular Diseases↗

PubMed1993

Biokinetics of technetium-99m-tetrofosmin: myocardial perfusion imaging agent: implications for a one-day imaging protocol.

Tetrofosmin is a 99mTc-labeled myocardial perfusion imaging agent that has shown encouraging results in Phase I and II clinical trials. The purpose of this study was to determine the biokinetics of this agent following administration during exercise and at rest in order to determine an optimal imaging protocol. Twenty patients with suspected coronary artery disease underwent symptom-limited treadmill exercise. Six to 8 mCi of 99mTc-tetrofosmin was injected at peak exercise and 22-24 mCi was injected 4 hr later at rest. Serial 5-min planar images were obtained in the left anterior oblique view at 5, 10, 15, 30, 60, 120 and 180 min after the radiotracer injection. Regions of interest were drawn on the serial images around the entire heart and portions of liver, lung, spleen, gallbladder and gastrointestinal tract. Average decay-corrected counts per pixel in each organ were plotted against time. In addition, heart-to-adjacent organ ratios were also determined. On stress images, the heart had the highest activity at all times, with the exception of gallbladder in the first 15 min. On rest images, the gallbladder, liver and gastrointestinal tract initially had higher activity than the heart; but the activity in these organs cleared rapidly over the subsequent 30-60 min. Heart-to-adjacent organ ratios were > 1.0 at all times in the stress images. Heart-to-organ ratios were < 1.0 in the first 15 min on the rest images for the liver and gastrointestinal tract. However, 30 min later, all ratios on the rest images were > or = 1.0. Technetium-99m-tetrofosmin images were considered to be of good to excellent quality with good myocardial delineation and adequate contrast between the heart and background. These These observations indicate that a convenient one-day tetrofosmin imaging protocol similar in duration to conventional 201Tl imaging is feasible.

Adult↗

PubMed2000

Two-dimensional electrophoretic analysis of Corynebacterium glutamicum membrane fraction and surface proteins.

An improved protocol for the two-dimensional analysis of proteins of the Corynebacterium glutamicum cytoplasmic membrane fraction is described. By use of increased 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) concentrations (2-4%) and an optimized electrophoresis protocol, horizontal streaking of proteins of the cytoplasmic membrane fraction was almost completely avoided. More important, in contrast to a previously published method, both a sample tray and IPG-phor isoelectric focusing unit can be used for the in-gel application of proteins. The described protocol was also found to be suitable for hydrophilic cytoplasmic proteins. Additionally, the preparation and analysis of C. glutamicum cell surface proteins is described. Proteins were extracted with lauroyl sarcosinate and 100-120 spots were separated on two-dimensional (2-D) gels in comparison to 18-20 spots observed previously by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). C. glutamicum proteins can now be separated into three distinct fractions resembling different functional units of the bacterial cell.

Amino Acid Sequence↗

PubMedDate not supplied

Optimization of techniques in screening CT of the sinuses.

The number of screening examinations of the sinuses performed with CT has markedly increased owing to the widespread and increasing use of endoscopic sinonasal surgery. We reviewed scans from 500 patients who had screening CT examinations of the sinuses for preendoscopic evaluation of inflammatory sinonasal disease to better define an optimal imaging protocol. Three aspects of direct coronal imaging of the paranasal sinuses were investigated: (1) preparation of the patient prior to the examination; (2) technical factors of the CT study, including positioning of the patient, optimal coronal angle, slice thickness, and CT exposure factors; and (3) data display. Our experience indicates that pretreatment of the patient with maximal medical therapy enables the best preendoscopic definition of anatomy, disease pattern, and nonreversible disease component for the treating surgeon. CT technical factors are optimized with scanning in the prone position with thin (3-mm) sections obtained through the anterior paranasal sinuses. This allows optimal visualization of the ostiomeatal unit. The remaining posterior portions of the sinuses are adequately imaged with thicker slices (5 mm). The coronal scan angle used is less critical. Exposure factors (mAs) can be reduced dramatically without image compromise. Data display is optimized when the bone algorithm is used to acquire the data and with image display at intermediate window center and width level. Use of the techniques outlined in this article results in a cost-effective yet diagnostic scan of the sinuses with decreased radiation exposure to the patient.

Cost-Benefit Analysis↗

PubMed1999

Lower esophageal sphincter dysfunction often precludes safe gastric feeding in stroke patients.

OBJECTIVES: To determine the relationship between stroke and lower esophageal dysfunction with vomiting and to identify an optimal nutrition protocol based on our findings. PATIENTS AND METHODS: The lower and upper esophageal sphincter functions were assessed in 35 patients who had an acute stroke to determine whether gastric or jejunal enteral feeding was the optimal route. Stroke was due to unilateral ischemia in 20 patients, unilateral intracerebral hemorrhage in 8 patients, and global ischemia in 7 patients. Our study consisted of 18 men and 17 women with an average age of 64 years. RESULTS: Using standard esophageal manometric definitions, the lower esophageal sphincter function was below normal in 24 patients: 3 had global anoxia, 5 had unilateral hemorrhage, and 16 had unilateral ischemia. The upper esophageal sphincter function was low in 30 patients: 6 had global anoxia, 7 had unilateral hemorrhage, and 17 had unilateral ischemia. Based on lower esophageal sphincter pressure, 7 patients underwent tube gastrostomy and 13 patients underwent tube jejunostomy placement. All tolerated enteral alimentation well. Prior to lower esophageal sphincter assessment, 4 patients had percutaneous endoscopy gastrostomy feedings that led to aspiration pneumonitis and consultation for tracheostomy; 2 terminally ill patients were referred to the ethics service, and 2 were converted to feeding via jejunostomy tube at the time of tracheostomy and did well. CONCLUSIONS: Vomiting with aspiration due to lower esophageal sphincter dysfunction is common after acute strokes. Esophageal manometry serves as a guide to find the optimal feeding route.

Adult↗

PubMed1995

High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBLs) are primary targets for gene therapy of inherited and acquired disorders of the immune system. We describe the development of an optimized transduction system that provides for high-efficiency retrovirus-mediated gene transfer into primary PBLs. This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low-temperature incubation (32 degrees C), and the use of the packaging cell line PG13. Gene marking studies of human and primate PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte population. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%. The transduction procedure does not alter phenotype, viability, or expansion of the transduced cells. Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which obviates the requirement for selection of transduced cells prior to gene-therapy procedures. Thus, large quantities of healthy retrovirally transduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a significant improvement in the methodology for the transduction of lymphocytes for gene therapy.

Animals↗

PubMed2026

Optimizing focal vibration therapy for balance and gait: A systematic review.

OBJECTIVE: This systematic review evaluated the efficacy of focal (localized) vibration therapy (FVT) applied to muscles/tendons on balance, gait, and mobility, with a specific focus on defining optimal vibration protocols (frequency, amplitude, dosing) and muscle-targeting strategies to maximize sensorimotor recovery. METHODS: A systematic review was conducted across six databases (CINHAL, Embase, Medline, Web of Science, Scopus, CENTRAL) from January 2000 to May 2025. Studies were included if they involved human participants, applied FVT therapeutically, and reported balance, gait, or mobility outcomes. Data extraction included study characteristics, intervention protocols, and outcomes. Methodological quality was assessed using the PEDro scale. RESULTS: Sixty-two studies (n = 2090 participants) were included. Methodological quality assessment (PEDro scale) indicated 44% of studies met high-quality standards. Biomechanical analysis identified the quadriceps, gastrocnemius/soleus, and plantar muscles as the most effective vibration sites, given their critical roles in gait propulsion and postural stability. The synthesis of protocol data indicated a promising therapeutic window characterized by a vibration frequency of 80-120 Hz (primarily fixed sinusoidal waveforms at a single frequency) and an amplitude of 0.2-0.5 mm (reported only in 12 studies; amplitude was not reported in 23 studies), applied bilaterally for a minimum of 3 sessions per week over 4-12 weeks, which could lead to improved balance and gait performance with benefits sustained for up to 5 months. CONCLUSION: FVT shows potential to improve gait and balance, particularly when targeting lower-extremity muscles with optimized vibration parameters. To advance the field, future research must prioritize the development of standardized protocols and investigate neurophysiological mechanisms to refine FVT as a precision bioengineering solution for mobility deficits.

Humans↗

PubMed2000

[Virtual endoscopy of the upper airway with spiral CT].

PURPOSE: The technical conditions, the optimized scanning protocols and the facilities of virtual endoscopy (VE) are presented in an overview. METHODS: Phantom studies showed that, for a single-row-detector helical CT, collimation of 3.0 mm combined with pitch of 1.5 provided an optimal compromise between image quality and the scan duration per breath-hold. A multi-row-detector helical CT requires only a fraction of the scanning time. This is especially important for patients with dyspnea. RESULTS: The threshold-dependent virtual endoscopic surface rendering is a reliable and rapidly practicable reconstruction algorithm for the imaging of the upper airway. CONCLUSIONS: The VE technique is suitable for the imaging of space-occupying tumors and restricted stenoses. With VE follow-up examinations can be performed non-invasively and interventional procedures can be prepared in an optimal way. Because of the computed tomographic data acquisition the structure of the mucosa is not assessable and there is no opportunity to obtain a biopsy specimen.

Bronchoscopes↗

PubMed1986

MR imaging of the lumbar spine: anatomic correlations and the effects of technical variations.

A correlative anatomic study, a retrospective review of MR images performed in 35 patients, and a series of tests of the effectiveness of various MR scanning techniques were performed in order to improve comprehension of lumbar spine anatomy depicted on MR images, and thereby facilitate development of an optimal scanning protocol. Correlation of MR images with cryomicrotomed cadaver specimens enhanced understanding of the MR depiction of the intervertebral disks, ligamentum flavum, nerve roots, epidural fat, and epidural veins. Experiments were performed to assess the efficacy of a surface coil applied to the back, a solenoidal surface coil, a standard body coil, and an abdominal compression device in optimizing image quality. Experiments were also performed to determine the effect of alterations in the pulse sequence and variations of the phase-encoding axis. Based on these results, a protocol is proposed for routine imaging of the lumbar spine that yields high-resolution sagittal and oblique images and that does not require a surface coil. The recommended protocol employs heavily T1-weighted images with phase encoding along the z axis for sagittal images and along the x axis for axial images. This protocol yields multiple sagittal and oblique axial images through each of the lumbar disks, a larger field of view than obtained with surface coils, and a reduction of total imaging time to as little as 10 min.

Cadaver↗

PubMedDate not supplied

Noncompetitive immunoassays using bifunctional unilamellar vesicles or liposomes.

Small unilamellar vesicles (SUVs) functionalized with an enzyme label and with specific ligands for biological molecules are useful as signal enhancement vehicles in the development of enzyme-linked immunoadsorbent assays and other biosensor applications. Bifunctional vesicles were prepared by covalently attaching horseradish peroxidase (HRP) and an antibody to the outside of the lipid bilayer of an SUV. The reaction conditions were optimized to obtain 7-12 antibody molecules and 100-200 HRP molecules per vesicle. The enzyme retained 70-80% of its specific activity after immobilization, and the presence of immobilized proteins on the vesicle surface apparently increased the vesicle stability. To minimize the background signal and maximize the specific signal, the immunoassay protocol was optimized with respect to (1) the type and concentration of blocking agent, (2) the diluents for HRP-antibody-vesicles and sample, (3) the incubation period, and (4) the incubation temperature. The bifunctional vesicles were used in a noncompetitive immunoassay to detect d-dimer, a fibrin dimer formed at the early stages of thrombosis. A second conjugate, HRP-antibody, was prepared, characterized, and used as a control against which to compare the assay using vesicles. The assay results using vesicles led to a detection limit for d-dimer in human plasma 9 times lower than what was achieved using the conventional enzyme-antibody conjugate assay.

Antibodies↗

PubMed1985

Theoretical considerations for optimizing intensity differences between primary musculoskeletal tumors and normal tissue with spin-echo magnetic resonance imaging.

In the radiographic assessment of primary musculoskeletal tumors, it is important for therapy planning to accurately define the extent of a tumor. Using a double spin-echo pulse sequence, the T1 and T2 relaxation times and relative hydrogen densities of several neoplastic tissues and of several normal tissues in four patients were measured. Neoplasms measured included one fibrosarcoma, two osteosarcomas, and one giant cell tumor. Normal tissues measured included normal muscle, fat, and bone marrow. Using a mathematical model of the double spin-echo pulse sequence, the intensity difference between each tumor and each normal tissue for multiple values of TR and TE was calculated. These calculated intensity differences were then used to plot isodifference contour curves for each tissue pair. These plots enabled us to pick combinations of TR and TE that optimized the signal difference between tumor and normal tissue. When comparing tumor with predominantly fatty tissue such as marrow or subcutaneous fat, optimal signal difference in our imager occurred at a TR of 600 to 800 msec and a very short TE. When comparing tumor with muscle, optimal signal difference occurred with very long TR times, and TE times ranging from 30 to 90 msec. These preliminary results suggest that an optimal scanning protocol for primary musculoskeletal tumors should contain at least two different pulse sequences with widely separated TR values (500 and 2000 msec in our instrument), and short to intermediate values of TE (28 and 56 msec in our instrument). It is believed that analysis of isodifference contour plots is a useful method for optimizing intensity differences between any two tissue types.

Bone Neoplasms↗

PubMed1994

Management of patients with acute asthma: what do we know? What do we need to know?

Management of patients with acute, severe asthma mandates the use of comprehensive monitoring and aggressive therapeutic drug regimens. Global clinical and objective assessment criteria can help determine the severity of the acute episode and will also help determine optimal management protocols. The response to aggressive therapy, rather than the initial severity of the attack, predicts outcome. Beta 2 sympathomimetic agents are the treatment of choice for acute asthmatic episodes. Aminophylline and parasympatholytic agents may be useful adjuvants, but more data are needed before firm conclusions can be reached regarding risk-benefit ratios. The optimal timing of steroid therapy is not yet known, but even this form of treatment is slow in producing results.

Acute Disease↗

PubMed2000

Outpatient parenteral inotropic therapy for advanced heart failure.

BACKGROUND: Patients with advanced heart failure generally have hemodynamic perturbation characterized by low cardiac output and high ventricular filling pressures. This creates a clinical milieu with profound symptomatology that includes weakness, fatigue, and fluid-retention states causing peripheral edema, mesenteric congestion, and dyspnea syndromes. Great morbidity including hospital admissions and readmissions as well as high mortality rates ensue. Though medication and/or surgical intervention often attenuate heart failure symptomatology, morbidity, and mortality, some patients reach more advanced stages despite aggressive maneuvers. Indeed, patients presenting with acute decompensation of chronic congestive heart failure frequently receive parenteral inotropic drugs during their hospitalization with clinical improvement. Because these agents generally increase cardiac output and reduce pre-load and afterload, they can be lifesaving. Some patients, however, have symptomatic and hemodynamic rebound to worsened heart failure states during or shortly after inotrope weaning. METHODS: It was, then, a logical step to segue from acute inpatient inotrope infusion to long-term administration of these drugs in the outpatient setting when patients were dependent on these agents. Dopamine, dobutamine, and milrinone are all generally available inotropes that have been used singly or in combination in a chronic outpatient infusion setting. CONCLUSIONS: Data from a few small clinical trials and anecdotal case experience suggest that these drugs result in both hemodynamic and clinical improvement that is generally sustained during chronic administration, and even noted long after discontinuation of infusions in some patients. Some reports have suggested that intermittent infusion therapy in outpatients (so-called pulsed therapy) is effective in attenuating congestive heart failure symptoms long term, with more data supporting chronic infusion of these agents. Though questions regarding safety of these agents have been raised, a reasonable compendium of data published to date supports the contention that inotropic drugs used in this fashion ameliorate symptoms. Legitimate concern may be raised regarding exacerbation of arrhythmias with subsequent sudden cardiac death syndrome; however, in severely symptomatic heart failure patients, the trade-off between symptomatic amelioration and the chance of sudden cardiac death may be worthwhile. Unfortunately, precise guidance regarding the best drug, dose, optimal administration technique, weaning protocol, and actual risk/benefit ratio are not well characterized. Practice as been guided, in large part, by anecdotal experience. However, it appears that chronic or pulsed outpatient parenteral inotropic infusion therapy is frequently prescribed and that this treatment option is an effective alternative for carefully selected patients with severely symptomatic and advanced heart failure. Formulating optimal protocols for home inotropic drug infusion therapy by conducting properly designed clinical trials will be an essential endeavor.

Adrenergic beta-Agonists↗

PubMed1987

Cryopreservation of in vitro cultured mouse preimplantation embryos.

We investigated two different freezing protocols on mouse embryos that were either grown in vivo or were grown in vitro for a certain period. Our results confirm that an extended in vitro culture period makes the embryo more susceptible for injury due to freezing and thawing. Furthermore when freezing two cell mouse embryos we could observe that this early stage is more dependent on optimal cryopreservation protocol parameters than later stages.

Animals↗

PubMed1982

Determination of half-dose depth in skin for soft x-rays.

Unlike superficial x-rays, the soft x-rays normally used in dermatologic practice spare unaffected underlying organs during treatment of cutaneous malignancies. However, since the dose with depth from soft x-rays varies markedly, it is important to know this relationship for optimal therapeutic results. The peak kilovoltage, and thus the energy of the beam, is generally selected so that the dose to the base of the lesion is one-half the surface dose. An absorbed dose of 3,400 rads to the surface and a dose of about one-half this amount to the base of most malignant lesions is one standard protocol for optimal therapeutic results. An accurate value of half-depth dose in skin is therefore necessary and is readily obtained from ordinary half-value layer measurements using the technic described.

Aluminum↗

PubMedDate not supplied

Detection of etiological agent for cholera by PCR protocol.

BACKGROUND: PCR protocol for Vibrio cholerae, the causative agent of the diarrheal disease cholera has been described in this report. We report the detection of Vibrio species in drinking water samples in a duplex PCR reaction. The target loci used in the study were ctxA and tcpA. The sensitivity and efficiency of detection of this protocol can be applied in epidemic conditions, wherein monitoring of target organisms is very crucial. MATERIAL AND METHODS: Single step thermocycling programs have been reported for amplification of specific target loci. We have demonstrated that a gradient multi-step thermocycling program is more efficient in improving the sensitivity of detection by PCR. The method for preparation of template DNA from environmental sample uses filtration of water followed by harvesting the possible bacterial residue. This was further treated with proteinase K and heat to yield DNA compatible for PCR. The protocol was optimized for amplification of target loci from standard strains as well as from environmental water samples. RESULTS: The method can simultaneously detect two different loci of Vibrio cholerae in a single reaction. The sensitivity of detection achieved for the pathogen was 100 cells per reaction. The specificity of the primers was demonstrated by spiking the reaction with non-specific template. The developed protocol was successfully extended to environmental samples. CONCLUSIONS: The developed duplex PCR protocol allows the simultaneous detection of two genetic loci of the target pathogen from water samples. This enhances the efficiency of detection and provides a sensitive tool for the rapid detection of the pathogen that can be useful in epidemic conditions where the time factor involved in the identification of target pathogen is very crucial.

Bacterial Outer Membrane Proteins↗