Resource-limited Laboratories Research Articles

Fast and low-cost patterning of electrodes on versatile 2D and 3D substrates by cutting and origami cohesive thermoplastic for biosensing applications

In this work, cohesive thermoplastic-assisted patterning was successfully used to fabricate versatile 2D and 3D electrodes for biosensing application. The cohesive thermoplastic template was fabricated by cutter shaping to remove wet-chemical processes and UV-lithography. This low cost approach is compatible with versatile inks and substrates. Both single and multi-step grafting of inks work well with cohesive thermoplastic templates to form electronic units on substrates. Other than work on a 2D flat substrate, the cohesive thermoplastic origami can easily fabricate electrodes on 3D objects, demonstrating itself as a good candidate to implant electronic unit on a stereotype object. The flexibility, convenience and operability seen here make this method a fast prototype method to evaluate the proof-of-concept design. It also provides alternative fabrication solutions for researchers from resource-limited laboratories to work on miniaturized devices.

A simple laboratory algorithm for diagnosis of melioidosis in resource-constrained areas: a study from north-central Vietnam

ObjectivesMelioidosis may be endemic in many tropical developing countries, but diagnosis of the disease is currently unreliable in resource-limited areas. We aimed to validate a simple and cheap laboratory algorithm for the identification of Burkholderia pseudomallei from clinical specimens in parts of Vietnam where the disease has not previously been reported. MethodsIn June 2015, we conducted training courses at five general hospitals in north-central provinces in order to raise awareness of the disease and to introduce a simple and cheap laboratory identification algorithm for B. pseudomallei including the three-antibiotic disc test. ResultsUntil the end of the year (7 months later), 94 suspected B. pseudomallei strains resistant to gentamicin and colistin but sensitive to amoxicillin/clavulanic acid were detected in clinical specimens from 70 patients. All strains were further confirmed as B. pseudomallei by using a specific TTSS1 real-time PCR assay and recA sequencing analysis. Among positive blood cultures, positive rates with B. pseudomallei ranged from 3.4% (5/147) to 10.2% (32/312) in the various clinics. A total of 82.8% (58/70) patients were bacteraemic, with a mortality of 50% (18/36) among patients with known outcome. No death occurred in nonbacteraemic patients. ConclusionsOur results demonstrate that the introduction of a simple and easy-to-perform laboratory algorithm for the identification of B. pseudomallei from clinical samples, together with clinical awareness raising, can lead to the diagnosis of a significant number of melioidosis cases in resource-limited clinical laboratories which previously did not identify the pathogen.

Molecular Serotype-specific Identification of Haemophilus influenzae Using the Loop-mediated Isothermal Amplification Method

BackgroundOver the past several decades, the incidence of meningitis caused by Haemophilus influenzae in children has decreased due to wide-spread vaccination of H. influenzae type b (Hib). The incidence of invasive diseases due to H. influenzae that were not included in the vaccines, however, has increased. We developed a rapid, simple, and cost-effective method for detecting serotypes of H. influenzae.MethodsWe designed LAMP primer sets based on the sequences available for the capsular types a, c, d, e, and f. The assays were evaluated regarding reactivity, specificity and sensitivity of the assays. To be used for the patients suspected meningitis, we evaluated the detection limit of the non-Hib serotype specific LAMP assay using DNAs spiking CSF specimens.ResultsThe reactivity and specificity of the LAMP assays were confirmed using 6 serotypes and nontype of H. influenzae strains, plus 21 strains representing other Haemophilus species and non-Haemophilus genera. The sensitivity of the LAMP assays was confirmed from 10 to 103 copies per reaction that was 1 to 103 times of sensitivity of the non-Hib serotype specific PCR that demonstrated the sensitivity 103 or 104 genome copies per reaction. The sensitivity of the LAMP was also the same, 10 to 103 genome copies per reaction.ConclusionThis is the first report of the serotype-specific identification assay for H. influenzaeusing the LAMP method. Our results suggest potential usage for children or adults with suspected meningitis in resource limited laboratories or public health surveillance systems.Funding sourceJSPS Bilateral Open Partnership Joint Research ProjectsDisclosures K. Takahashi, Kaneka Corp.: Employee, Salary; S. Hayakawa, Kaneka Corp.: Collaborator, Research grant

Detection of T and B cells specific complement-fixing alloantibodies using flow cytometry: A diagnostic approach for a resource limited laboratory.

BACKGROUND AND OBJECTIVES:Various methods have been reported for the detection of antibodies in recipient sera, which can be human leukocyte antigens (HLAs) or non-HLA specific, complement- or noncomplement fixing, as well as donor T (HLA-Class-I) and/or B cell (HLA-Class-I and II) specific. These alloantibodies play a pivotal role in antibody-mediated renal transplantation rejection. Deposition of C4d in peritubular capillaries of a kidney biopsy is a marker of antibody-mediated rejection. The C4d flow-panel reactive antibodies (PRAs) are a screening method for HLA-specific and complement fixing antibodies. However, the method is limited by the lack of donor specificity.DESIGN AND SETTINGS:Here, we present a new and simple flow cytometric method referred to as C4d-flow cytometry crossmatch (C4d-FCXM) for the detection of donor-specific (T and/or B cell) and C4d-fixing alloantibodies.RESULTS:The method was applied in a series of clinical cases and judged to be useful. The method may limit unwanted deferral of the donor due to positivity in C4d Flow-PRA and/or FCXM and may be helpful in prediction of antibody mediated rejections. Furthermore, this method can provide information pretransplant in contrast to kidney biopsy and C4d evaluation done posttransplant.CONCLUSIONS:We postulate that this method incorporates most of the features of all the available modalities (i.e., National Institute of Health-complement dependent lymphocytotoxicity, FCXM, cytotoxic FCXM and C4d-flowPRA) yet cost-effective and best suited for resource-limited laboratory/ies which is a common scenario in developing countries.

Thermal desorption modulation for comprehensive two-dimensional gas chromatography using a simple and inexpensive segmented-loop fluidic interface

In this study, we introduce a modulation strategy for comprehensive two-dimensional gas chromatography (GC×GC) by using a simple and consumable-free modulator. This “Do-It-Yourself” interface comprised a 1.0m×0.25mm segment of MTX-5 metallic column and a low-cost DC power supply. Thermal desorption modulation (TDM) was attained using a dual-stage heater-based modulator in a novel segmented-loop configuration. TDM was achieved by alternating analyte trapping and thermal desorption. Former process relied on analyte partition to sorbent phase, while latter explored direct resistive heating. Introduction of an intermediate delay segment between the two stages mitigated analyte breakthrough, improving peak symmetry and chromatographic efficiency. This feature was critical to acquire reliable GC×GC modulation using such simple heater-based device. The effects of important modulation variables on 2D separations were investigated, including TDM stage length, dimension of delay loop, and outlet pressure. Significant advances and limitations of proposed TDM strategy were carefully determined. Proposed GC×GC prototype by using an in-oven TDM modulator was successfully applied to a series of challenging matrices, including petroleum distillates, biodiesel, and essential oil. This open-hardware, cost-effective modulator was easy to install and operate, as it circumvented the need for sophisticated components (e.g. moving parts and cooling systems). Therefore, our modulator is a compelling alternative to existing GC×GC solutions to operate in resource-limited laboratories.

Facial fabrication of paper-based flexible electronics with flash foam stamp lithography

Paper-based flexible electronics/circuit have a bright potential in displays, transistor and sensors due to low cost and disposability. Here we presented a facile microfabrication method for paper-based flexible electronics with flash foam stamp lithography (FFSL), which can be implemented in resource-limited laboratory. Only two steps are needed. Firstly, negative patterns of circuit are stamped on the paper with polydimethylsiloxane (PDMS) ink to form hydrophobic barriers after PDMS is solidified. Then the patterned paper is immersed in the conductive ink to absorb ink and acquire circuits. In this paper we present foldable, waterproof and multi-layer paper-based flexible electronics can be easily fabricated. Also as FFSL can be also directly used in fabrication of microfluidic paper-based analytical devices (μPADs), this method has a promising potential in fabricating electrodes of μPADs.

Proficient Detection of Multi-Drug-Resistant Mycobacterium tuberculosis by Padlock Probes and Lateral Flow Nucleic Acid Biosensors.

Tuberculosis is a major communicable disease. Its causative agent, Mycobacterium tuberculosis, becomes resistant to antibiotics by acquisition of point mutations in the chromosome. Multi-drug-resistant tuberculosis (MDR-TB) is an increasing public health threat, and prompt detection of such strains is of critical importance. As rolling circle amplification of padlock probes can be used to robustly distinguish single-nucleotide variants, we combined this technique with a sensitive lateral flow nucleic acid biosensor to develop a rapid molecular diagnostic test for MDR-TB. A proof-of-concept test was established for detection of the most common mutations [rpoB 531 (TCG/TTG) and katG 315 (AGC/ACC)] causing MDR-TB and verification of loss of the respective wild type. The molecular diagnostic test produces visual signals corresponding to the respective genotypes on lateral flow strips in approximately 75 min. By detecting only two mutations, the test can detect about 60% of all MDR-TB cases. The padlock probe-lateral flow (PLP-LF) test is the first of its kind and can ideally be performed at resource-limited clinical laboratories. Rapid information about the drug-susceptibility pattern can assist clinicians to choose suitable treatment regimens and take appropriate infection control actions rather than prescribing empirical treatment, thereby helping to control the spread of MDR-TB in the community.

Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation.

Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories.

Fast prototyping of a customized microfluidic device in a non-clean-room setting by cutting and laminating Parafilm®

Cutting and laminating Parafilm® film to build versatile microfluidic chips provides a solution for resource-limited laboratories to conduct microfluidic study.

A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7

E. coli O157:H7 is one of the most important foodborne pathogen that causes some human illnesses such as bloody diarrhea, hemolytic-uremic syndrome, and kidney failure. We developed a loop-mediated isothermal amplification (LAMP) assay with six special primers that target a highly specific 299-bp region of the Z3276 gene for the detection of E. coli O157:H7. Among 117 bacterial strains tested in this study, positive results were only obtained from E. coli O157:H7 strains. The sensitivity level of the Z3276-LAMP assay was determined to be 5 CFU/reaction tube in pure bacterial culture. Moreover, the LAMP assay was successfully applied to artificially contaminated ground beef with a sensitivity level of 103 CFU/mL without pre-enrichment and 10 CFU/mL after a 4-h pre-enrichment. In conclusion, the present LAMP assay would be a useful and powerful tool for the rapid, sensitive, and specific diagnosis of E. coli O157:H7 strains in resource limited laboratories.

Measurement of Cholesterol Sub-Fractions, High Density Lipoprotein 2 and High Density Lipoprotein 3 in Type 2 Diabetes Mellitus Patients in Burkina Faso (West Africa)

According to literature, the High Density Lipoprotein 2 (HDL2) cholesterol is a better predictor of cardiovascular disease than total HDL. Thus, this study was undertaken to evaluate the interest of HDL2 in the management of Type 2 Diabetes Mellitus (T2DM) patients recruited at University Hospital Yalgado Ouedraogo of Ouagadougou (Burkina Faso, West Africa).The distribution of type 2 diabetes group according to cardiovascular complications reported 37.9% with microvascular complications and 36.4% with macrovascular complications. The total cholesterol, triglyceride, HDL cholesterol and HDL3 cholesterol were significantly higher in type 2 diabetes population compared to control group (p<0.001). On the contrary, a significant decrease of HDL2 cholesterol and LDL cholesterol were observed in T2DM group compared to control group (p<0.05). The decrease of HDL2 cholesterol according to the cardiovascular risk factors was significant in obesity, diabetes duration up to 10 years and hypercholesterolemia. The HDL2 cholesterol variations according to diabetes complications showed significant decrease in metabolic syndrome and hypertension (p<0.001). The HDL2 cholesterol measurement was useful for evaluating cardiovascular risk in diabetics, because in this study only its level was significantly decreased, while total cholesterol and HDL3 cholesterol levels increased and LDL cholesterol level was within normal limits. Therefore, the HDL2 might be including in T2DM management. Indeed, the reagents are available from local providers and the quality control has validated this affordable method for resource limited laboratories.

Genotypic assessment of drug-resistant tuberculosis in Baghdad and other Iraqi provinces using low-cost and low-density DNA microarrays.

We report on a molecular investigation carried out to ascertain the prevalence of drug-resistant tuberculosis (TB) and the specific gene mutations responsible for resistance to rifampicin (RIF) and/or isoniazid (INH) in Iraq. In total, 110 clinical isolates from category II TB cases from Baghdad (58%) and several Iraqi provinces (42%) were analysed using colorimetric, low-cost and low-density (LCD) microarrays (MYCO-Direct and MYCO-Resist LCD array kits) to identify the point mutations responsible for resistance in Mycobacterium tuberculosis isolates. We found 76 patients (69.1%) had resistant strains, of which 40 (36%) were multidrug-resistant (MDR)-TB. Where mono-resistance was identified, it was found to be predominantly to RIF (83%). The most common mutations were rpoB S531L (50%), inhA C15T (25%) and katG S315T (15%). The most common MDR-TB genotypes were rpoB S531L with inhA C15T (60%) and rpoB S531L with katG S315T (20%). Where phenotypic analysis of clinical isolates was also performed, genotypic data were found to show excellent correlation with phenotypic results. Correlation was found between the MYCO-Resist LCD array and GenoType MTBDRplus for detection of resistance to RIF. Our study shows MDR-TB in 36% of category II TB cases in Baghdad and surrounding Iraqi provinces, which reflects the World Health Organization findings based on phenotypic studies. Diagnosis of TB and MDR-TB using culture-based tests is a significant impediment to global TB control. The LCD arrays investigated herein are easy to use, sensitive and specific molecular tools for TB resistance profiling in resource-limited laboratory settings.

CYP2B6 poor metaboliser alleles involved in efavirenz and nevirapine metabolism: CYP2B6*9 and CYP2B6*18 distribution in HIV-exposed subjects from Dschang, Western Cameroon

The prescription of patients’ tailored anti-infectious treatments is the ultimate goal of pharmacogenetics/genomics applied to antimicrobial treatments, providing a basis for personalized medicine. Despite the efforts to screen Africans for alleles underlying defective metabolism for a panel of different drugs, still more research is necessary to clarify the interplay between host genetic variation and treatments’ response. HIV is a major infectious disease in sub-Saharan African countries, and the main prescribed anti-HIV combination therapy includes efavirenz (EFV) or nevirapine (NVP). The two drugs are both mainly metabolised by cytochrome P450 2B6 liver enzyme (CYP2B6). Defective variants of CYP2B6 gene, leading to higher drug exposure with subsequent possible side effects and low compliance, are well known. However, little is known about CYP2B6 alleles in Cameroon where only one study was done on this subject. The main objective of the present work is to assess, in a subset of HIV-exposed subjects from Dschang in West Cameroon, the prevalence of two SNPs in the CYP2B6 gene: 516G>T (rs3745274) and 983T>C (rs28399499), both associated to a defective EFV and NVP metabolism. We analyzed 168 DNA samples collected during two cross-sectional surveys performed in Dschang, West Cameroon. In the population studied the observed allele frequencies of 516G>T and 983T>C were 44.35% (95%CI, 36.84–51.86%) and 12.80% (95%CI, 7.75–17.85%), respectively. Moreover, concerning the CYP2B6 expected phenotypes, 28.57% of the population showed a poor metaboliser phenotype, while 27.38% and 44.05% showed an extensive (wild-type) and an intermediate metaboliser phenotype, respectively. Here we found that an important fraction of the subjects is carrying EFV/NVP poor metaboliser alleles. Our findings could help to improve the knowledge about the previewed efficacy of anti-HIV drug therapy in Cameroon. Finally, we designed a new method of detection for the 983T>C genetic variation that can be applied in resource-limited laboratories.

A flexible, small positron emission tomography prototype for resource-limited laboratories

Modern small-animal PET scanners typically consist of a large number of detectors along with complex electronics to provide tomographic images for research in the preclinical sciences that use animal models. These systems can be expensive, especially for resource-limited educational and academic institutions in developing countries. In this work we show that a small-animal PET scanner can be built with a relatively reduced budget while, at the same time, achieving relatively high performance. The prototype consists of four detector modules each composed of LYSO pixelated crystal arrays (individual crystal elements of dimensions 1 × 1 × 10 mm3) coupled to position-sensitive photomultiplier tubes. Tomographic images are obtained by rotating the subject to complete enough projections for image reconstruction. Image quality was evaluated for different reconstruction algorithms including filtered back-projection and iterative reconstruction with maximum likelihood-expectation maximization and maximum a posteriori methods. The system matrix was computed both with geometric considerations and by Monte Carlo simulations. Prior to image reconstruction, Fourier data rebinning was used to increase the number of lines of response used. The system was evaluated for energy resolution at 511 keV (best 18.2%), system sensitivity (0.24%), spatial resolution (best 0.87 mm), scatter fraction (4.8%) and noise equivalent count-rate. The system can be scaled-up to include up to 8 detector modules, increasing detection efficiency, and its price may be reduced as newer solid state detectors become available replacing the traditional photomultiplier tubes. Prototypes like this may prove to be very valuable for educational, training, preclinical and other biological research purposes.

Loop-mediated isothermal amplification for diagnosis of 18 World Organization for Animal Health (OIE) notifiable viral diseases of ruminants, swine and poultry.

Loop-mediated isothermal amplification (LAMP) is a simple, powerful state-of-the-art gene amplification technique used for the rapid diagnosis and early detection of microbial diseases. Many LAMP assays have been developed and validated for important epizootic diseases of livestock. We review the LAMP assays that have been developed for the detection of 18 viruses deemed notifiable of ruminants, swine and poultry by the World Organization for Animal Health (OIE). LAMP provides a fast (the assay often takes less than an hour), low cost, highly sensitive, highly specific and less laborious alternative to detect infectious disease agents. The LAMP procedure can be completed under isothermal conditions so thermocyclers are not needed. The ease of use of the LAMP assay allows adaptability to field conditions and works well in developing countries with resource-limited laboratories. However, this technology is still underutilized in the field of veterinary diagnostics despite its huge capabilities.

Fast and Accurate Identification of &amp;lt;i&amp;gt;M. tuberculosis&amp;lt;/i&amp;gt; Complex Using an Immunochromatographic MPT64 Antigen Detection Test

Background: A new rapid Immunochromatographic test (ICT) kit (MPT64 TB Ag Kit) for detection of MPT64 Antigen in M. tuberculosis (MTB) isolates used for rapid identification of MTB isolates developed by SD (Standard Diagnostics) Bio line, South Korea was evaluated. The ICT is a rapid, reliable and cheaper method that can be used instead of conventional biochemical tests for confirming MTB in culture isolates in resource limited laboratories. The study also evaluated the ability of ICT to detect MPT64-Antigen before the micro MGIT could signal positive. Material/Methods: A total of 450 sputum samples of individual patients were used for the study. 152 isolates of Mycobacteria were recovered from solid and liquid media. These strains were tested for the detection of MPT64-antigen. H37Rv strain was served as the positive reference control and also used for early detection of Antigen experiment. Findings: The development of bands on both test and sample region when H37Rv strain was tested were seen (MPT64 antigen positive). When 138 MTB isolates were tested, it showed a similar banding pattern indicating 100% sensitivity. MPT64 band formation was not detected in any of the 14 isolates indicating 100% specificity. Both PPV & NPV were 100%. All the isolates negative for MPT64 Ag were confirmed as MOTT by conventional bio-chemical PNBA. The H37Rv strain showed a faint band from the 2nd day onwards from inoculation till 3rd day in the earlier Antigen detection experiment. Conclusion: Rapid identification of MTB culture isolate is a pressing need for diagnosis and proceeding to perform drug susceptibility testing. MPT64 TB Ag detection ICT kit is a rapid, reliable method, good substitute for molecular identification methods, and conventional biochemical test which is time-consuming and technically demanding. The early detection of Antigen can be used as an effective tool in diagnosis.

A rapid manual processing technique for resource-limited small laboratories.

Background:Surgical pathology is an integral part of diagnosis and management planning in patient care. In the absence of widespread automation, many small laboratories are unable to provide this service due to lack of time. Currently, the shortest processing schedule for formalin-fixed paraffin-embedded tissues is 16 working hours; most small laboratories cannot complete the rapid schedules within the average 8–9 working hours. Thus, the availability of an 8–9 h processing schedule that provide satisfactory results can help many small laboratories in routinely providing surgical pathology services.Objectives:To evaluate the effectiveness of a new rapid processing schedule and compare it with two existing rapid processing schedules.Materials and Methods:This animal study tested a new rapid processing schedule suggested by authors with overnight preprocessing in 60% isopropyl alcohol followed by an 8 h processing schedule. This was tested and compared with the rapid processing schedules described by Godkar's (11 h) and Bancroft's (2 working days). A routinely used automatic tissue processor long cycle (17 h) was the control. Each schedule was used on 20 tongue specimens. The prepared slides were evaluated for surface area and linear tissue shrinkage, ease of sectioning, quality of hematoxylin and eosin staining, histological appearance and artifacts.Results:No statistical differences were found between schedules. Overall total average performance ranking placed Bancroft's schedule as the best with only 27.9% of the sections processed and stained showing any shortcomings, followed closely by the test schedule suggested by authors (28.7%), Godkar's (31.8%) and the automatic processor schedule/control (33.3%).Conclusion:The test results indicated that the schedule devised by authors is an effective rapid processing cycle that produces diagnostic quality histological results when compared with other conventional processing schedules for small tissue blocks (average 6 mm × 8 mm).

One-post patterning of multiple protein gradients using a low-cost flash foam stamp.

Printing of versatile chemical and biological inks for protein and cell patterning was achieved using a simple and cost-effective flash foam stamp (FFS). The grey-scale mask fabricated stamp can generate multiple protein gradients with one-post stamping. Due to the importance of spatially controlled protein patterns in both biology and tissue engineering, this straightforward and reliable tool is an accessible solution for resource-limited laboratories conducting molecular patterning experiments.

Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation -assay-based diagnostic kit

Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates −80°C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5M sucrose–PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37°C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. These results confirm the viability of a freeze-dried PVNA-based kit, which could significantly facilitate low-cost serology for a wide portfolio of emerging infectious viruses.

An in silico model for rapid identification of multiple bacteria in resource limited laboratories.

We have developed an in silico model for simplified detection of five major food borne bacteria using common PCR primer based on conserved region flanking a variable region in the first rRNA operon sequences. The RFLP pattern exhibited on virtual SmaI and MboI digestion were unique to each genus. This design is based on the combination of comparative sequence analysis, conventional PCR and restriction digestion methods. We believe this approach as a better alternative to 16S rRNA sequencing based identification / detection of bacteria and suited for the resource limited academic research laboratories in which variety of bacteria are used in different students' projects.