scholarly journals Polymerase Chain Reaction Multiplication for the Detection of Bacterial Isolates Causing Neonatal Sepsis

2020 ◽  
Vol 8 (A) ◽  
pp. 623-628
Author(s):  
Utari Hartati Suryani ◽  
Andri Rezano ◽  
Yunia Sribudiani
Keyword(s):  
Polymerase Chain Reaction ◽  
Neonatal Sepsis ◽  
Pcr Analysis ◽  
Local Alignment ◽  
Bacterial Isolates ◽  
Chain Reaction ◽  
E Coli ◽  
Initial Design ◽  
Pcr Multiplex ◽  
Polymerase Chain

BACKGROUND: Neonatal sepsis is a clinical syndrome caused by the presence of bacteria in the blood accompanied by symptoms and systemic signs of infection that occurs in the first 4 weeks of life after birth. The process of identifying pathogenic microorganisms is essential in determining the clinical condition in neonatal sepsis. AIM: The study was aimed to develop a multiplex polymerase chain reaction (PCR) method to identify bacterial isolates that cause neonatal sepsis in Indonesia with the main target of optimization of an initial design and PCR optimization. METHODS: This research is an explorative in vitro study for the optimization of an initial design and PCR methods for the detection of the main bacteria that cause sepsis neonatorum in Indonesia, namely, bacteria Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, and Pseudomonas aeruginosa. The study was conducted at the Biomolecular Laboratory of the Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia. RESULTS: Sequencing carried out and continued with Basic Local Alignment Search Tool (BLAST) sequencing results, it appears that the PCR product that has been produced conforms with the optimization targets that were previously set when doing the primer design. The level of homology found in all four species based on the results of BLAST in a sequence is as follows: K. pneumoniae 94%, P. aeruginosa 99%, E. cloacae 100%, and E. coli 100%. CONCLUSION: PCR multiplex method using design primers and conventional PCR analysis methods (using agarose gel) can be used to detect four species of bacteria that cause neonatal sepsis, namely, K. pneumoniae, P. aeruginosa, E. cloacae, and E. coli.

Evaluation of Methods To Improve Detection of Escherichia coli O157:H7 in Fresh Produce by Multiplex Polymerase Chain Reaction

2003 ◽  
Vol 66 (1) ◽  
pp. 18-24 ◽  
Author(s):  
MICHAEL A. GRANT
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Fresh Produce ◽  
Nucleic Acid Amplification ◽  
Detection Sensitivity ◽  
Pcr Analysis ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

Multiplex polymerase chain reaction (PCR) analysis was used to detect two genes encoding Shiga-like toxins (stx1 and stx2) and a universal Escherichia coli gene (gadA/B) in fresh produce spiked with E. coli O157:H7. Current U.S. Food and Drug Administration procedures for the analysis of fresh produce include the use of the rinsate from an initial rinse for the analysis of several potential pathogens, including E. coli O157:H7. In this study, several procedures were evaluated for their ability to increase the sensitivity of PCR analysis of rinsates from 15 types of produce. The procedures evaluated included the preliminary clarification and concentration of templates by centrifugation and the treatment of templates with compounds reported to facilitate nucleic acid amplification, including polyvinlypolypyrrolidone (PVPP), nonfat dry milk (NFDM), and InstaGene. The preliminary concentration of rinsates resulted in moderate improvements in detection sensitivity. The use of PVPP-treated templates in PCR reaction mixtures did not further improve sensitivity, but the inclusion of NFDM-treated templates increased sensitivity by an order of magnitude for 12 rinsates. The incorporation of InstaGene also improved the detection capability of the analysis; this procedure yielded the strongest gel bands for eight rinsates. However, for four other rinsates, the use of this reagent decreased sensitivity; these four rinsates were those for the produce varieties with the largest surface areas and were the most turbid rinsates. The use of facilitating compounds to block PCR inhibition may enable an analysis for Shiga toxin–producing E. coli in fresh produce to be completed in 1 to 2 days, rather than the 5 days required for current methods.

Sensitivity of polymerase chain reaction (PCR)-southern hybridization and conventional PCR analysis for Halal authentication of gelatin capsules

LWT ◽  
2015 ◽  
Vol 63 (1) ◽  
pp. 714-719 ◽  
Author(s):  
Sahilah Abd Mutalib ◽  
Nursheila Mustafa Muin ◽  
Aminah Abdullah ◽  
Osman Hassan ◽  
Wan Aida Wan Mustapha ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Southern Hybridization ◽  
Pcr Analysis ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Gelatin Capsules ◽  
Halal Authentication ◽  
Polymerase Chain

Development of Three-Stage Bioaerosol Sampler for Size-Selective Sampling

2022 ◽  
Author(s):  
Jun-Hyung Lim ◽  
Sang Hwan Nam ◽  
Jongwoo Kim ◽  
Nam Hoon Kim ◽  
Gun-Soo Park ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Size Range ◽  
Collection Efficiency ◽  
Commercial Product ◽  
Pcr Analysis ◽  
Cyclone Separator ◽  
Chain Reaction ◽  
Selective Sampling ◽  
Sampling Flow Rate ◽  
Polymerase Chain

Abstract In this study, a three-stage bioaerosol sampler with a sampling flow rate of 170 L/min was designed and fabricated for sampling the bioaerosols released during human breathing and coughing, and its performance was evaluated. The sampler was constructed using a cyclone separator with a cutoff size of 2.5 µm as a preseparator, a multi-nozzle virtual impactor with a cutoff size of 0.34 µm as an aerosol concentrator, and a BioSampler, which is a commercial product, for collecting bioaerosols in a collection fluid. The collection efficiency of the sampler was evaluated through simulations and experiments. Only particles with sizes of 0.1-4 µm were selectively collected in the collection fluid. Bacteriophage bioaerosols were sampled using the developed sampler and ACD-200 Bobcat sampler, which is a commercial product. The amounts of collected bacteriophages were compared using the polymerase chain reaction (PCR) technique. The sampling performance of the developed sampler was similar to that of the ACD-200 Bobcat sampler. Moreover, the developed sampler showed its ability to sample bioaerosols of a specific size-range and collect them directly in a collection fluid for the PCR analysis. Therefore, the developed sampler is expected to be useful for indoor environmental monitoring by effectively sampling the bioaerosols released indoors during human breathing and coughing.

scholarly journals Occurrence of toxigenic Escherichia coli in raw milk cheese in Brazil

2007 ◽  
Vol 59 (2) ◽  
pp. 508-512 ◽  
Author(s):  
B.R. Paneto ◽  
R.P. Schocken-Iturrino ◽  
C. Macedo ◽  
E. Santo ◽  
J.M. Marin
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Antimicrobial Agents ◽  
Raw Milk ◽  
Diffusion Method ◽  
Chain Reaction ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Polymerase Chain ◽  
Raw Milk Cheese

The occurrence of toxigenic Escherichia coli in raw milk cheese was surveyed in Middle Western Brazil. Fifty samples of cheese from different supermarkets were analyzed for E.coli. The isolates were serotyped and screened for the presence of verotoxigenic E. coli (VTEC) and enterotoxigenic E. coli (ETEC) by Polymerase Chain Reaction (PCR). The susceptibility to thirteen antimicrobial agents was evaluated by the disk diffusion method. E.coli were recovered from 48 (96.0%) of the samples. The serogroups identified were O125 (6.0%), O111 (4.0%), O55 (2.0%) and O119 (2.0%). Three (6.0%) and 1(2.0%) of the E.coli isolates were VTEC and ETEC, respectively. Most frequent resistance was observed to the following antimicrobials: cephalothin (60.0%), nalidixic acid (40.0%), doxycyclin (33.0%), tetracycline (31.0%) and ampicillin (29.0%).

scholarly journals Application of Polymerase Chain Reaction for the Correlation of Salmonella Serovars Recovered from Greyhound Feces with Their Diet

1993 ◽  
Vol 5 (3) ◽  
pp. 378-385 ◽  
Author(s):  
Gregory G. Stone ◽  
M. M. Chengappa ◽  
Richard D. Oberst ◽  
Nathan H. Gabbert ◽  
Scott McVey ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Fecal Material ◽  
Chain Reaction ◽  
Antigenic Analysis ◽  
Fecal Samples ◽  
E Coli ◽  
Staphylococcus Intermedius ◽  
Standard Procedures ◽  
Polymerase Chain ◽  
Salmonella Serovars

The polymerase chain reaction was employed to correlate Salmonella serovars isolated from fecal material of greyhounds suffering from gastroenteritis with those isolated from the diet fed to the greyhounds prior to onset of diarrhea. Kennels around the Abilene, Kansas, area were contacted and supplied with materials needed to collect a portion of the diet each day. With t e onset of diarrhea, the kennels were instructed to ship the fecal material and diet from the previous 10 days to the laboratory for testing. Forty-one fecal samples and corresponding diets were screened for Salmonella, Clostridium perfringens, Campylobacterjejuni, Staphylococcus aureus, Staphylococcus intermedius, and pathogenic (piliated) Escherichia coli by direct culture using standard procedures. The fecal material was also screened for coronavirus and parvovirus using electron microscopy. Thirty-five “normal” fecal samples were screened for all of the above mentioned microorganisms as a control. In addition, the fecal material was screened for E. coli verotoxins I and II and clostridial enterotoxins. A total of 61 Salmonella isolates were recovered from the 41 samples of feces and diet submitted for testing; 31 were recovered from the feces and 30 from the diet. Four Salmonella isolates were recovered from the normal fecal samples. Results obtained by PCR, plasmid profiles, antigenic analysis, and antibiogram profiles indicated that 16 of the 31 isolates recovered from the fecal material were the same strain as that recovered from the diet.

scholarly journals Multiplex Polymerase Chain Reaction Assay for Identification of Enterotoxigenic Escherichia Coli Strains

2001 ◽  
Vol 13 (4) ◽  
pp. 308-311 ◽  
Author(s):  
Jacek Osek
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Direct Determination ◽  
Enteric Bacteria ◽  
Enterotoxigenic Escherichia Coli ◽  
Chain Reaction ◽  
Gram Negative ◽  
E Coli ◽  
Polymerase Chain

A multiplex polymerase chain reaction (PCR) system was developed for identification of enterotoxigenic Escherichia coli (ETEC) strains and to differentiate them from other gram negative enteric bacteria. This test simultaneously amplifies heat-labile (LTI) and heat-stable (STI and STII) toxin sequences and the E. coli-specific universal stress protein ( uspA). The specificity of the method was validated by single PCR tests performed with the reference E. coli and non- E. coli strains and with bacteria isolated from pig feces. The multiplex PCR allowed the rapid and specific identification of enterotoxin-positive E. coli and may be used as a method for direct determination of ETEC and to differentiate them from other E. coli and gram-negative enteric isolates.

Quantitative PCR Analysis for Bcl-2/IgH in a Phase III Study of Yttrium-90 Ibritumomab Tiuxetan As Consolidation of First Remission in Patients With Follicular Lymphoma

2009 ◽  
Vol 27 (36) ◽  
pp. 6094-6100 ◽  
Author(s):  
Lindsey Goff ◽  
Karin Summers ◽  
Sameena Iqbal ◽  
Jens Kuhlmann ◽  
Michael Kunz ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Follicular Lymphoma ◽  
Quantitative Polymerase Chain Reaction ◽  
Phase Iii ◽  
Pcr Analysis ◽  
Control Group ◽  
Ibritumomab Tiuxetan ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Yttrium 90

Purpose The randomized First-Line Indolent Trial (FIT) was conducted in patients with advanced follicular lymphoma (FL), to evaluate the safety and efficacy of yttrium-90 (90Y) ibritumomab tiuxetan given as consolidation of complete or partial remission. This study of minimal residual disease was undertaken in parallel, to determine the rate of conversion from bcl-2 polymerase chain reaction (PCR) –detectable to –undetectable status and the corresponding effect on progression-free survival (PFS). Patients and Methods Blood samples from 414 patients (90Y-ibritumomab, n = 208; control, n = 206) were evaluated using real-time quantitative polymerase chain reaction (RQ-PCR); 186 were found to have the bcl-2 rearrangement and were thus eligible for inclusion in the RQ-PCR analysis. Results Overall, 90% of treated patients converted from bcl-2 PCR–detectable to –undetectable disease status, compared with 36% in the control group. Treatment significantly prolonged median PFS in patients converting to bcl-2 PCR-undetectable status (40.8 v 24.0 months in the control group; P < .01, hazard ratio [HR], 0.399). In patients who had bcl-2 PCR-detectable disease at random assignment, treatment significantly prolonged median PFS (38.4 v 8.2 months in the control group; P < .01, HR, 0.293). Conclusion Eradication of PCR-detectable disease occurred more frequently after treatment with 90Y-ibritumomab tiuxetan and was associated with prolongation of PFS.

scholarly journals Polymerase Chain Reaction and blood culture for diagnosis of canine sepsis

2018 ◽  
Vol 48 (6) ◽  
Author(s):  
Marcelo Marques da Silveira ◽  
Stéfhano Luis Cândido ◽  
Karin Rinaldi dos Santos ◽  
Maerle Oliveira Maia ◽  
Roberto Lopes de Souza ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Culture ◽  
Diagnostic Value ◽  
Turnaround Time ◽  
Diagnostic Techniques ◽  
Pcr Analysis ◽  
Chain Reaction ◽  
Suspected Sepsis ◽  
Fungal Dna ◽  
Polymerase Chain

ABSTRACT: Sepsis is characterized by the presence of organ dysfunction secondary to the dysregulated systemic inflammatory response associated with an infection, and has high mortality rates. Traditional diagnostic techniques based on non-microbiological isolation are time-consuming and may delay treatment. Thus, this study aimed to compare bacterial and fungal broad-range polymerase chain reaction (PCR) and blood culture for diagnosis of sepsis in dogs. Blood samples from 88 dogs with suspected sepsis were analyzed by blood culture, and PCR to detect bacterial and fungal DNA. On blood culture, 20 (22.7%) samples tested positive for bacterial isolates; however, none tested positive for fungi. Through PCR analysis, bacterial DNA was detected in 46 (52.3%) animals, whereas fungal DNA was present in one (1.1%) sample. Our results showed that PCR-based testing has important diagnostic value for canine blood infections because it has a shorter turnaround time and higher sensitivity than traditional blood culture.

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