scholarly journals Direct Detection of Streptococcus suis from Cerebrospinal Fluid, Positive Hemoculture, and Simultaneous Differentiation of Serotypes 1, 1/2, 2, and 14 within Single Reaction

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 996
Author(s):  
Ingyin Shun Lae Thu ◽  
Khajornsak Tragoolpua ◽  
Sorasak Intorasoot ◽  
Usanee Anukool ◽  
Phadungkiat Khamnoi ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Direct Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Streptococcus Suis ◽  
Perfect Agreement ◽  
Efficient Treatment ◽  
Multiplex Pcr Assay ◽  
Routine Identification ◽  
Single Reaction

Streptococcus suis is an emerging zoonotic bacterium causing septicemia and meningitis in humans. Due to rapid disease progression, high mortality rate, and many underdiagnosed cases by time-consuming routine identification methods, alternative diagnostic testing is essential. Among 29 broadly accepted S. suis serotypes, serotypes 2 and 14 are high prevalent; however, many PCR assays showed an inability to differentiate serotype 2 from 1/2, and 1 from 14. In this study, we developed and validated a new multiplex PCR assay that facilitates the identification of only the 29 true serotypes of S. suis and simultaneously differentiates serotypes 1, 1/2, 2, and 14 within a single reaction. Importantly, the multiplex PCR could detect S. suis directly from positive hemocultures and CSF. The results revealed high sensitivity, specificity, and 100% accuracy with almost perfect agreement (κ = 1.0) compared to culture and serotyping methods. Direct detection enables a decrease in overall diagnosis time, rapid and efficient treatment, reduced fatality rates, and proficient disease control. This multiplex PCR offers a rapid, easy, and cost-effective method that can be applied in a routine laboratory. Furthermore, it is promising for developing point-of-care testing (POCT) for S. suis detection in the future.

scholarly journals Outcome of Colonization of Apis mellifera by Nosema ceranae

2007 ◽  
Vol 73 (20) ◽  
pp. 6331-6338 ◽  
Author(s):  
Raquel Martín-Hernández ◽  
Aránzazu Meana ◽  
Lourdes Prieto ◽  
Amparo Martínez Salvador ◽  
Encarna Garrido-Bailón ◽  
...  
Keyword(s):  
Apis Mellifera ◽  
Multiplex Pcr ◽  
Small Subunit ◽  
Nosema Ceranae ◽  
Small Subunit Rrna ◽  
Specific Detection ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Year 2000 ◽  
Single Reaction

ABSTRACT A multiplex PCR-based method, in which two small-subunit rRNA regions are simultaneously amplified in a single reaction, was designed for parallel detection of honeybee microsporidians (Nosema apis and Nosema ceranae). Each of two pairs of primers exclusively amplified the 16S rRNA targeted gene of a specific microsporidian. The multiplex PCR assay was useful for specific detection of the two species of microsporidians related to bee nosemosis, not only in purified spores but also in honeybee homogenates and in naturally infected bees. The multiplex PCR assay was also able to detect coinfections by the two species. Screening of bee samples from Spain, Switzerland, France, and Germany using the PCR technique revealed a greater presence of N. ceranae than of N. apis in Europe, although both species are widely distributed. From the year 2000 onward, statistically significant differences have been found in the proportions of Nosema spp. spore-positive samples collected between and within years. In the first period examined (1999 to 2002), the smallest number of samples diagnosed as Nosema positive was found during the summer months, showing clear seasonality in the diagnosis, which is characteristic of N. apis. From 2003 onward a change in the tendency resulted in an increase in Nosema-positive samples in all months until 2005, when a total absence of seasonality was detected. A significant causative association between the presence of N. ceranae and hive depopulation clearly indicates that the colonization of Apis mellifera by N. ceranae is related to bee losses.

scholarly journals Development of a Single-Reaction Multiplex PCR Toxin Typing Assay for Staphylococcus aureusStrains

2000 ◽  
Vol 66 (4) ◽  
pp. 1347-1353 ◽  
Author(s):  
Naresh K. Sharma ◽  
Catherine E. D. Rees ◽  
Christine E. R. Dodd
Keyword(s):  
Multiplex Pcr ◽  
Toxin Gene ◽  
Gene Products ◽  
Pcr Assay ◽  
Specific Primers ◽  
Purification Method ◽  
Multiplex Pcr Assay ◽  
Enterotoxin Genes ◽  
Immunological Tests ◽  
Single Reaction

ABSTRACT We describe here the development of a single-reaction multiplex PCR assay for the enterotoxin genes from Staphylococcus aureusthat utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products. In combination with a new DNA purification method, the assay can detect enterotoxin genes A to E from a pure culture within 3 to 4 h. The test was used to characterize a diverse set of environmental S. aureus isolates, and a 99% correlation with toxin typing using standard immunological tests was found. The design of the assay allows it to be extended to include both newly characterized and as-yet-unknown toxin genes.

scholarly journals A SARS-CoV-2 Coronavirus Antigen-Detecting Half-Strip Lateral Flow Assay Towards the Development of Point of Care Tests Using Commercially Available Reagents

2020 ◽  
Author(s):  
Benjamin D. Grant ◽  
Caitlin E. Anderson ◽  
John R Williford ◽  
Luis F. Alonzo ◽  
Veronika A. Glukhova ◽  
...  
Keyword(s):  
Point Of Care ◽  
Diagnostic Testing ◽  
Lateral Flow ◽  
Rt Pcr ◽  
Middle Income ◽  
Efficient Treatment ◽  
Clinical Sensitivity ◽  
Starting Point ◽  
Half Strip ◽  
Implementation Costs

<p>The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clinically sensitive, may fill many of the gaps in the current RT-PCR testing regime, especially in low- and middle-income countries (LMICs). To date, many serology LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, based on previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clinical sensitivity than serology assays. To date, only a very small number of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this paper we present a half-strip LFA using commercially available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.62 ng/mL using an optical reader with sensitivity equivalent to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests. </p>

Direct Detection and Identification of Arcobacter Species by Multiplex PCR in Chicken and Wastewater Samples from Spain

2007 ◽  
Vol 70 (2) ◽  
pp. 341-347 ◽  
Author(s):  
A. GONZÁLEZ ◽  
S. BOTELLA ◽  
R. M. MONTES ◽  
Y. MORENO ◽  
M. A. FERRÚS
Keyword(s):  
Multiplex Pcr ◽  
Water Samples ◽  
Direct Detection ◽  
Pcr Amplification ◽  
Pcr Assay ◽  
Direct Pcr ◽  
Culture Methods ◽  
Multiplex Pcr Assay ◽  
Detection And Identification ◽  
Wastewater Samples

Twenty-two chicken livers, 10 chicken carcasses, and 15 wastewater samples were processed and analyzed for Arcobacter by PCR and traditional culture methods. Samples were enriched for 24 and 48 h, incubated at 30°C under aerobic conditions, and streaked on blood selective media. To determine the best isolation conditions, 20 samples also were processed under microaerophilic conditions at 37°C. Simple and multiplex PCR assays were used directly with enrichment broths and isolated strains. Seventeen Arcobacter strains were isolated from chicken samples, and A. butzleri was the only Arcobacter species identified. The direct PCR assay revealed that 29 of the 32 chicken samples were contaminated with Arcobacter. A. butzleri was the most frequently detected species, although Arcobacter cryaerophilus also was present in some of the samples and Arcobacter skirrowii occasionally was detected. All the wastewater samples were positive by PCR assay for Arcobacter after 24 h of enrichment. A. butzleri and A. cryaerophilus were detected with the multiplex PCR assay. Fourteen Arcobacter strains were isolated from 10 of the 15 water samples analyzed; 7 were identified as A. butzleri and the remaining 7 were A. cryaerophilus. Both for chicken and water samples, Arcobacter detection rate for PCR amplification was higher than for culture isolation. These results indicate the high prevalence of Arcobacter in chicken and wastewater and the inadequacy of available cultural methods for its detection. The species-specific multiplex PCR assay is a rapid method for assessing Arcobacter contamination in chicken and wastewater samples and is a viable alternative to biochemical identification of isolated strains.

scholarly journals Direct Detection and Identification of the Most Common Bacteria and Fungi Causing Otitis Externa by a Stepwise Multiplex PCR

2021 ◽  
Vol 11 ◽  
Author(s):  
Shima Aboutalebian ◽  
Kazem Ahmadikia ◽  
Hamed Fakhim ◽  
Javaher Chabavizadeh ◽  
Ahmadreza Okhovat ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Otitis Externa ◽  
Fungal Pathogens ◽  
Direct Detection ◽  
Specific Primers ◽  
Multiplex Pcr Assay ◽  
Detection And Identification ◽  
Amplicon Size ◽  
Bacteria And Fungi ◽  
Species Specific

BackgroundConsidering the importance of differential diagnosis of infectious otitis externa (OE), a stepwise PCR-based assay using universal and genus- or species-specific primers for the detection/identification of the most prevalent bacterial and fungal OE was developed and evaluated on the ear aspiration specimens of clinically suspected patients.Methods and MaterialsA total of 120 ear aspiration specimens with otomycosis suspicion were subjected to manual DNA extraction using phenol–chloroform extraction after tissue digestion with a lysis buffer. The multiplex PCR was initially performed using pan-fungal and bacterial homemade primers. Pseudomonas and Staphylococcus specific primers were simultaneously used in one reaction mixture to identify the bacterial genera. Furthermore, for the identification of fungal agents, Candida species-specific multiplex primers targeting the most clinically important Candida species causing OE (i.e., C. albicans, C. parapsilosis, and C. auris), as well as Aspergillus related multiplex PCR identifying the most prevalent Aspergillus species were used in two separate reaction mixtures. All the results of multiplex PCR were interpreted based on the amplicon size.ResultsThe overall multiplex PCR-based detection rate of bacterial (n = 88; 73.3%) and fungal (n = 97; 81%) OE was documented to be 100% along with and complete consistency with the results of direct examination and Giemsa staining. Double amplicon bands of bacterial and fungal pathogens were evidenced in 76 specimens (63.3%). Moreover, the positivity rate of pan-fungal PCR was higher than that of the culture result. Out of 88 pan-bacterial positive PCR specimens, 66 and 47 ones were positive for Staphylococcus and Pseudomonas, respectively. In addition, 30 samples exhibited mixed infection of both, and five specimens remained negative. Out of 97 pan-fungal positive PCR specimens, 67 and 51 ones contained Candida and Aspergillus species, respectively. It should be noted that dual amplicon bands of Candida and Aspergillus-related multiplex PCR were yielded in 30 specimens.ConclusionThe stepwise multiplex PCR assay proved to be more sensitive, more rapid, as well as less cumbersome in detection and identification of fungal and bacterial OE, compared to culture.

scholarly journals Characterization of the Immunoglobulin Heavy- and Light-Chain Repertoires in a Single Reaction

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-14
Author(s):  
Geoffrey Lowman ◽  
Michelle Toro ◽  
Jayde Chang ◽  
Loni Pickle ◽  
Chenchen Yang ◽  
...  
Keyword(s):  
B Cell ◽  
Multiplex Pcr ◽  
Cell Line ◽  
Light Chain ◽  
Conflicts Of Interest ◽  
Limit Of Detection ◽  
Oncology Research ◽  
Multiplex Pcr Assay ◽  
Clonality Assessment ◽  
Single Reaction

Background B cell repertoire analysis by next-generation sequencing (NGS) has shown particular utility in the field of hematological oncology research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to flow-based methods, and the elimination of specifically designed primers often required for qPCR-based methods. Owing to primer-primer interactions and incompatibility of reaction conditions, current multiplex PCR assays require separate PCR reactions to survey each immunoglobulin chain (IGH, IGK, IGL), often leading to a longer time-to-answer for samples in which no marker is initially detected. We have developed an assay for receptor analysis based on Ion AmpliSeq technology to circumvent these issues, allowing the effective use of up to thousands of primers in a single reaction. The highly multiplexed, pan-clonality NGS assay provides for efficient detection of IGH, IGK, and IGL chain rearrangements in a single reaction. Methods We developed a single primer panel targeting the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IGK, IGL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IGK loci rearrangements involving Kappa deletion and C intron elements. To evaluate performance, we conducted clonality assessment and limit-of-detection testing used gDNA from a total of 45 research samples representing common B cell malignancies. We included samples derived from peripheral blood, bone marrow, and FFPE-preserved tissues at input levels ranging from 100ng to 2µg. Finally, we further characterized the samples via a separate AmpliSeq-based multiplex PCR assay targeting rearranged TCRB and TCRG chains. Sequencing and clonality analysis was performed using the Ion GeneStudio S5 System and Ion Reporter 5.16. Results Clonality assessments carried out using gDNA collected from both cell line and clinical research samples (CLL, B-ALL, Multiple Myeloma, Burkitt's Lymphoma, NHL, and DLBCL) show a &gt;90% overall positive detection rate. Assessment of linearity-of-response and limit-of-detection was carried out using cell lines diluted in PBL to between 10-3 and 10-6 by mass. The multi-receptor assay performs as expected, with linear response to the cell line frequency across the range tested, including the ability to detect clones of interest at 10-6. Conclusions These results demonstrate the robustness of our newly developed Ion AmpliSeq-assay for B cell receptor heavy and light chains. We expect this assay to simplify the workflow for clonality assessment and rare clone detection in B cell malignancy research. For research use only. Disclosures No relevant conflicts of interest to declare.

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