scholarly journals Thermostable Heptaplex PCR Assay for the Detection of Six Respiratory Bacterial Pathogens

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 753
Author(s):  
Nik Mohd Noor Nik Zuraina ◽  
Mohammed Dauda Goni ◽  
Khazani Nur Amalina ◽  
Habsah Hasan ◽  
Suharni Mohamad ◽  
...  
Keyword(s):  
Multiplex Polymerase Chain Reaction ◽  
Bacterial Pathogens ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Base Pairs ◽  
Specific Primers ◽  
Taq Dna Polymerase ◽  
Simple Detection ◽  
Polymerase Chain ◽  
Mpcr Assay

A thermostabilized, multiplex polymerase chain reaction (mPCR) assay was developed in this study for the detection of six respiratory bacterial pathogens. Specific primers were designed for an internal amplification control (IAC) and six target sequences from Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Haemophilus influenzae. The resultant seven-band positive amplification control (PAC) of this heptaplex PCR assay corresponded to 105 base pairs (bp) of IAC, 202 bp of K. pneumoniae, 293 bp of S. aureus, 349 bp of S. pneumoniae, 444 bp of P. aeruginosa, 505 bp of M. tuberculosis, and 582 bp of H. influenzae. Results found that 6% (w/v) of the stabilizer was optimum to preserve the functional conformation of Taq DNA polymerase enzyme. This assay was stable at ambient temperature for at least 6 months. The sensitivity and specificity of this assay were both 100% when testing on the intended target organisms (n = 119) and non-intended species (n = 57). The mPCR assay developed in this study enabled accurate, rapid, and simple detection of six respiratory bacteria.

scholarly journals Application of a multiplex polymerase chain reaction (mPCR) assay to detect fraud by substitution of bovine meat cuts with water buffalo meat in Northern Brazil

2019 ◽  
Vol 17 (1) ◽  
pp. 790-795 ◽  
Author(s):  
Vanderson Vasconcelos Dantas ◽  
Gabrielle Virginia Ferreira Cardoso ◽  
Wanessa Shuelen Costa Araújo ◽  
Andrey Carlos do Sacramento de Oliveira ◽  
Andreia Silva da Silva ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Water Buffalo ◽  
Chain Reaction ◽  
Buffalo Meat ◽  
Northern Brazil ◽  
Polymerase Chain ◽  
Mpcr Assay

scholarly journals Application of a Multiplex Polymerase Chain Reaction Assay for the Simultaneous Confirmation of Listeria monocytogenes and Other Listeria Species in Turkey Sample Surveillance†

2002 ◽  
Vol 65 (5) ◽  
pp. 780-785 ◽  
Author(s):  
IRENE V. WESLEY ◽  
KAREN M. HARMON ◽  
JAMES S. DICKSON ◽  
ANN RAMOS SCHWARTZ
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Listeria Species ◽  
Multiplex Pcr Assay ◽  
Polymerase Chain

A multiplex polymerase chain reaction was developed to simultaneously identify Listeria monocytogenes and species of the genus Listeria. Two sets of primers were used, with the first amplifying a 938-bp region of the 16S rRNA gene that is highly conserved in all Listeria species and the second amplifying a 174-bp region of the listeriolysin (hlyA) gene of L. monocytogenes. Thus, isolates of Listeria spp. yield a single 938-bp product, whereas L. monocytogenes isolates yield both the 938-bp product and a 174-bp product. The specificity of the assay was verified with all six Listeria species and 11 serotypes of L. monocytogenes, as well as nonrelated bacteria. The multiplex PCR assay was used to determine the incidence of Listeria spp., especially L. monocytogenes, in mechanically separated turkey samples (n = 150 samples). L. monocytogenes strains were selected by using the University of Vermont two-step enrichment protocol and plating to selective Palcam agar. The multiplex PCR assay was used for verification of presumptive Listeria colonies. Approximately 38% of mechanically separated turkey samples (57 of 150) yielded L. monocytogenes; an additional 18% of these samples (27 of 150) harbored other Listeria spp. Fifty-one percent (29 of 57) of the L. monocytogenes isolates were of serogroup 1, 44% (25 of 57) were of serogroup 4, and 2% (1 of 57) were assigned to serogroups other than 1 and 4.

scholarly journals 1774. Impact of a Multiplex Polymerase Chain Reaction Assay on the Clinical Management of Adults Undergoing a Lumbar Puncture for Suspected Community-Onset Central Nervous System Infections

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S654-S654
Author(s):  
Matthew Moffa ◽  
Rawiya Elrufay ◽  
Thomas L Walsh ◽  
Dustin R Carr ◽  
Nathan Shively ◽  
...  
Keyword(s):  
Multiplex Polymerase Chain Reaction ◽  
Intervention Group ◽  
Turnaround Time ◽  
Cns Infection ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Post Intervention ◽  
Multiplex Pcr Assay ◽  
Polymerase Chain ◽  
Post Intervention Group

Abstract Background Patients admitted from the community with a suspected central nervous system (CNS) infection require prompt antimicrobial treatment and diagnostic evaluation. Our health network recently implemented a multiplex polymerase chain reaction (PCR) assay in-house. Methods This was a pre-/post-intervention study evaluating the impact that a multiplex PCR assay had on the clinical management of patients ≥18 years of age admitted from the community with a lumbar puncture (LP) performed for a suspected CNS infection. The primary outcome was Herpes Simplex Virus (HSV) PCR turnaround time (TAT). Secondary outcomes included inpatient length of stay (LOS), total antimicrobial days of therapy (DOT), and antiviral DOT. Patients were excluded if an LP was performed after hospital day 3, if they were on a systemic antimicrobial for a non-CNS indication, if they were a neurosurgical patient, and if they had a fungal CNS infection. Results The pre- and post-intervention groups each had 57 patients. The average age was 51 and 52 years in the pre- and post-intervention groups, respectively. Four patients (7%) in the pre-intervention group were immunocompromised, compared with 9 (16%) in the post-intervention group. Four patients in the pre-intervention group had a positive PCR assay for either HSV or Varicella Zoster Virus (VZV), compared with 5 patients in the post-intervention group. Neither group had a positive cerebrospinal fluid culture, bacterial antigen assay, or bacterial PCR assay. The median (IQR) HSV PCR TAT was significantly longer in the pre-intervention group, 85 (78, 96) vs. 3.9 hours (2.9, 4.7), P < 0.001. The mean LOS was numerically greater in the pre-intervention arm (7 vs. 4.7 days, P = 0.069), as were the total antimicrobial DOT (9 vs. 7.4 days, P = 0.279) and antiviral DOT (3.9 vs. 2.7 days, P = 0.136). Pre-intervention antiviral DOT was significantly greater (3.1 vs. 1.6 days, P = 0.011) in patients without a positive HSV or VZV PCR. Conclusion Implementing a multiplex PCR assay for adults undergoing an LP for a suspected CNS infection significantly reduced the HSV PCR turnaround time. Antiviral DOT was significantly shorter in patients with a negative PCR result post-intervention. We also found a non-significant reduction in LOS, total antimicrobial DOT, and antiviral DOT. Disclosures All authors: No reported disclosures.

Multiplex polymerase chain reaction as a mastitis screening test for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis in bulk milk samples

2003 ◽  
Vol 70 (2) ◽  
pp. 149-155 ◽  
Author(s):  
Patchara Phuektes ◽  
Glenn F Browning ◽  
Garry Anderson ◽  
Peter D Mansell
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Streptococcus Dysgalactiae ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Milk Samples ◽  
Streptococcus Uberis ◽  
Bulk Milk ◽  
Polymerase Chain ◽  
Total Bacteria

Effective diagnostic tools for screening herds for mastitis pathogens are important in development and monitoring of mastitis control programmes. A multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis was used in preliminary studies to assess its applicability as an alternative method for monitoring mastitis caused by these organisms at the herd level. PCR was used to detect the presence of these organisms in bulk milk samples. Correlations with bulk milk somatic cell counts (BMCC), total bacteria counts and thermoduric bacteria counts were evaluated. A total of 176 bulk milk samples were collected from 42 herds on five consecutive occasions at approx. 10-d intervals. Str. uberis was the most common organism in these bulk milk samples. There was no relationship between presence of either Staph. aureus, Str. dysgalactiae or Str. uberis and BMCC, total bacteria counts or thermoduric bacteria counts. However, presence of Str. agalactiae was associated with high BMCC and total bacteria counts. The results of this study show that regular analysis of bulk milk using this multiplex PCR assay may be a useful tool for monitoring herd status with respect to Str. agalactiae, but is of less value for monitoring occurrence of Staph. aureus, Str. dysgalactiae and Str. uberis. Further investigations are needed to clarify the relationship between positive PCR results and the prevalence of infected cows in the herd.

scholarly journals Application of RT-PCR for Indexing Avocado Sunblotch Viroid

Plant Disease ◽  
1997 ◽  
Vol 81 (9) ◽  
pp. 1023-1026 ◽  
Author(s):  
R. J. Schnell ◽  
D. N. Kuhn ◽  
C. M. Ronning ◽  
D. Harkins
Keyword(s):  
Rt Pcr ◽  
Amplification Product ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Specific Primers ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Dideoxynucleotide Chain Termination Method ◽  
Slab Gels ◽  
Avocado Sunblotch Viroid

A method for the routine detection of avocado sunblotch viroid (ASBVd) in nucleic acid extracts of infected avocado tissues by reverse transcription-polymerase chain reaction (RT-PCR) was developed using ASBVd-specific primers. Amplified cDNA products were analyzed by electrophoresis on nondenaturing 6% polyacrylamide slab gels. The size of the major RT-PCR product from ASBVd-infected tissue was estimated to be 250 bp. This product was absent from amplified extracts of uninfected tissue. The amplification product from ASBVd was sequenced by the dideoxynucleotide chain termination method, and the sequence was over 97% identical to the published sequence. The RT-PCR assay is sensitive enough to allow viroid detection without requiring large amounts of tissue, highly purified ASBVd, or molecular hybridization.

scholarly journals Detection of pathogenicYersinia enterocoliticausing the multiplex polymerase chain reaction

1996 ◽  
Vol 117 (1) ◽  
pp. 59-67 ◽  
Author(s):  
N. Harnett ◽  
Y. P. Lin ◽  
C. Krishnan
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Oligonucleotide Probes ◽  
Chain Reaction ◽  
Base Pairs ◽  
Single Colony ◽  
Colony Forming Units ◽  
The Family ◽  
Stool Specimens ◽  
Polymerase Chain

SummaryA multiplex polymerase chain reaction (PCR) was developed to detect the presence of theail, yst, andvirFgenes ofYersinia enterocoliticasimultaneously, quickly and accurately. The amplified fragment sizes were 356 base-pairs (bp) for theailgene, 134 bp for theystgene, and 231 bp for thevirFgene. The specificity of the amplified products was confirmed by hybridization with digoxigenin-labelled oligonucleotide probes. Amplification was successful whether the template was derived from a single colony of bacteria, aliquots of boiled bacterial suspensions, from DNA extracted from pure or mixed cultures or from stool specimens. Amplification of thevirFgene was also achieved from strains ofY. pseudotuberculosiscarrying the 70 kb plasmid but not with preparations from other relatedYersiniaspecies or from other members of the familyEnterobacteriaceae. The detection limit we established was 5–10 colony forming units per millilitre (cfu/ml) and 1·0 pg of DNA.

scholarly journals Simultaneous detection of major blackleg and soft rot bacterial pathogens in potato by multiplex polymerase chain reaction

2014 ◽  
Vol 165 (3) ◽  
pp. 474-487 ◽  
Author(s):  
M. Potrykus ◽  
W. Sledz ◽  
M. Golanowska ◽  
M. Slawiak ◽  
A. Binek ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Bacterial Pathogens ◽  
Simultaneous Detection ◽  
Soft Rot ◽  
Chain Reaction ◽  
Polymerase Chain
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