scholarly journals Multiplex Droplet Digital Polymerase Chain Reaction Assay for Rapid Molecular Detection of Pathogens in Patients With Sepsis: Protocol for an Assay Development Study

10.2196/33746 ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. e33746
Author(s):  
Samir Badran ◽  
Ming Chen ◽  
John E Coia
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Culture ◽  
Sensitivity And Specificity ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Phase 3 ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Background Blood cultures are the cornerstone of diagnosis for detecting the presence of bacteria or fungi in the blood, with an average detection time of 48 hours and failure to detect a pathogen occurring in approximately 50% of patients with sepsis. Rapid diagnosis would facilitate earlier treatment and/or an earlier switch to narrow-spectrum antibiotics. Objective The aim of this study is to develop and implement a multiplex droplet digital polymerase chain reaction (ddPCR) assay as a routine diagnostic tool in the detection and identification of pathogens from whole blood and/or blood culture after 3 hours of incubation. Methods The study consists of three phases: (1) design of primer-probe pairs for accurate and reliable quantification of the most common sepsis-causing microorganisms using a multiplex reaction, (2) determination of the analytical sensitivity and specificity of the multiplex ddPCR assay, and (3) a clinical study in patients with sepsis using the assay. The QX200 Droplet Digital PCR System will be used for the detection of the following species-specific genes in blood from patients with sepsis: coa (staphylocoagulase) in Staphylococcus aureus, cpsA (capsular polysaccharide) in Streptococcus pneumoniae, uidA (beta-D-glucuronidase) in Escherichia coli, oprL (peptidoglycan-associated lipoprotein) in Pseudomonas aeruginosa, and the highly conserved regions of the 16S rRNA gene for Gram-positive and Gram-negative bacteria. All data will be analyzed using QuantaSoft Analysis Pro Software. Results In phase 1, to determine the optimal annealing temperature for the designed primer-probe pairs, results from a gradient temperature experiment will be collected and the limit of detection (LOD) of the assay will be determined. In phase 2, results for the analytical sensitivity and specificity of the assay will be obtained after an optimization of the extraction and purification method in spiked blood. In phase 3, clinical sensitivity and specificity as compared to the standard blood culture technique will be determined using 301 clinical samples. Conclusions Successful design of primer-probe pairs in the first phase and subsequent optimization and determination of the LOD will allow progression to phase 3 to compare the novel method with existing blood culture methods. International Registered Report Identifier (IRRID) PRR1-10.2196/33746

scholarly journals Multiplex Droplet Digital Polymerase Chain Reaction Assay for Rapid Molecular Detection of Pathogens in Patients With Sepsis: Protocol for an Assay Development Study (Preprint)

2021 ◽  
Author(s):  
Samir Badran ◽  
Ming Chen ◽  
John E Coia
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Culture ◽  
Sensitivity And Specificity ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Phase 3 ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

BACKGROUND Blood cultures are the cornerstone of diagnosis for detecting the presence of bacteria or fungi in the blood, with an average detection time of 48 hours and failure to detect a pathogen occurring in approximately 50% of patients with sepsis. Rapid diagnosis would facilitate earlier treatment and/or an earlier switch to narrow-spectrum antibiotics. OBJECTIVE The aim of this study is to develop and implement a multiplex droplet digital polymerase chain reaction (ddPCR) assay as a routine diagnostic tool in the detection and identification of pathogens from whole blood and/or blood culture after 3 hours of incubation. METHODS The study consists of three phases: (1) design of primer-probe pairs for accurate and reliable quantification of the most common sepsis-causing microorganisms using a multiplex reaction, (2) determination of the analytical sensitivity and specificity of the multiplex ddPCR assay, and (3) a clinical study in patients with sepsis using the assay. The QX200 Droplet Digital PCR System will be used for the detection of the following species-specific genes in blood from patients with sepsis: <i>coa</i> (staphylocoagulase) in <i>Staphylococcus aureus</i>, <i>cpsA</i> (capsular polysaccharide) in <i>Streptococcus pneumoniae</i>, <i>uidA</i> (beta-D-glucuronidase) in <i>Escherichia coli</i>, <i>oprL</i> (peptidoglycan-associated lipoprotein) in <i>Pseudomonas aeruginosa</i>, and the highly conserved regions of the 16S rRNA gene for Gram-positive and Gram-negative bacteria. All data will be analyzed using QuantaSoft Analysis Pro Software. RESULTS In phase 1, to determine the optimal annealing temperature for the designed primer-probe pairs, results from a gradient temperature experiment will be collected and the limit of detection (LOD) of the assay will be determined. In phase 2, results for the analytical sensitivity and specificity of the assay will be obtained after an optimization of the extraction and purification method in spiked blood. In phase 3, clinical sensitivity and specificity as compared to the standard blood culture technique will be determined using 301 clinical samples. CONCLUSIONS Successful design of primer-probe pairs in the first phase and subsequent optimization and determination of the LOD will allow progression to phase 3 to compare the novel method with existing blood culture methods. INTERNATIONAL REGISTERED REPORT PRR1-10.2196/33746

scholarly journals Evaluation of Amplification Targets for the Specific Detection ofBordetella pertussisUsing Real-Time Polymerase Chain Reaction

2014 ◽  
Vol 25 (4) ◽  
pp. 217-221 ◽  
Author(s):  
Mohammad Rubayet Hasan ◽  
Rusung Tan ◽  
Ghada N Al-Rawahi ◽  
Eva Thomas ◽  
Peter Tilley
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Target Genes ◽  
Reference Panel ◽  
Superior Performance ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

BACKGROUND:Bordetella pertussisinfections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR) assays to detectB pertussisare typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity.METHODS: A novelB pertussisreal-time PCR assay based on the porin gene was tested in parallel with several previously published assays that target genes such as IS481,ptx-promoter, pertactin and a putative thialase. The assays were evaluated using a reference panel of common respiratory bacteria including differentBordetellaspecies and 107 clinical nasopharyngeal specimens. Discrepant results were confirmed by sequencing the PCR products.RESULTS: Analytical sensitivity was highest for the assay targeting the IS481element; however, the assay lacked specificity forB pertussisin the reference panel and in the clinical samples. False-positive results were also observed with assays targeting theptx-promoter and pertactin genes. A PCR assay based on the thialase gene was highly specific but failed to detect all reference strains ofB pertussis. However, a novel assay targeting the porin gene demonstrated high specificity forB pertussisboth in the reference panel and in clinical samples and, based on sequence-confirmed results, correctly predicted allB pertussis-positive cases in clinical samples. According to Probit regression analysis, the 95% detection limit of the new assay was 4 colony forming units/reaction.CONCLUSION: A novel porin assay forB pertussisdemonstrated superior performance and may be useful for improved molecular detection ofB pertussisin clinical specimens.

Polymerase chain reaction for the amplification of the 121-bp repetitive sequence ofSchistosoma mansoni: a highly sensitive potential diagnostic tool for areas of low endemicity

2014 ◽  
Vol 89 (6) ◽  
pp. 769-773 ◽  
Author(s):  
E. Ferrer ◽  
F. Pérez ◽  
I. Bello ◽  
A. Bolívar ◽  
M. Lares ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Sensitivity And Specificity ◽  
Repetitive Sequence ◽  
Annealing Temperature ◽  
Immunological Methods ◽  
Molecular Technique ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Highly Sensitive ◽  
Polymerase Chain

AbstractSchistosomiasis is a disease caused by parasitic flatworms of the genusSchistosoma, whose diagnosis has limitations, such as the low sensitivity and specificity of parasitological and immunological methods, respectively. In the present study an alternative molecular technique requiring previous standardization was carried out using the polymerase chain reaction (PCR) for the amplification of a 121-bp highly repetitive sequence forSchistosoma mansoni.DNA was extracted from eggs ofS. mansoniby salting out. Different conditions were standardized for the PCR technique, including the concentration of reagents and the DNA template, annealing temperature and number of cycles, followed by the determination of the analytical sensitivity and specificity of the technique. Furthermore, the standardized PCR technique was employed in DNA extracted, using Chelex®100, from samples of sera of patients with an immunodiagnosis of schistosomiasis. The optimal conditions for the PCR were 2.5 mmMgCl2, 150 mmdeoxynucleoside triphosphates (dNTPs), 0.4 μmprimers, 0.75 U DNA polymerase, using 35 cycles and an annealing temperature of 63°C. The analytical sensitivity of the PCR was 10 attograms of DNA and the specificity was 100%. The DNA sequence was successfully detected in the sera of two patients, demonstrating schistosomiasis transmission, although low, in the community studied. The standardized PCR technique, using smaller amounts of reagents than in the original protocol, is highly sensitive and specific for the detection of DNA fromS. mansoniand could be an important tool for diagnosis in areas of low endemicity.

scholarly journals A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay With Enhanced Capacity to Detect Vesicular Stomatitis Viral Lineages of Central American Origin

2021 ◽  
Vol 8 ◽  
Author(s):  
Kate Hole ◽  
Charles Nfon ◽  
Luis L. Rodriguez ◽  
Lauro Velazquez-Salinas
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Polymerase Chain Reaction Assay ◽  
Detection Sensitivity ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Vesicular Stomatitis ◽  
Polymerase Chain

Vesicular stomatitis virus (VSV) causes a disease in susceptible livestock that is clinically indistinguishable from foot-and-mouth disease. Rapid testing is therefore critical to identify VSV and rule out FMD. We previously developed and validated a multiplex real-time reverse transcription polymerase chain reaction assay (mRRT-PCR) for detection of both VS New Jersey virus (VSNJV) and VS Indiana virus (VSIV). However, it was subsequently apparent that this assay failed to detect some VSNJV isolates in Mexico, especially in genetic group II, lineage 2.1. In order to enhance the sensitivity of the mRRT-PCR for VSNJV, parts of the assay were redesigned and revalidated using new and improved PCR chemistries. The redesign markedly improved the assay by increasing the VSNJV detection sensitivity of lineage 2.1 and thereby allowing detection of all VSNJV clades. The new assay showed an increased capability to detect VSNJV. Specifically, the new mRRT-PCR detected VSNJV in 100% (87/87) of samples from Mexico in 2006-2007 compared to 74% for the previous mRRT-PCR. Furthermore, the analytical sensitivity of the new mRRT-PCR was enhanced for VSNJV. Importantly, the modified assay had the same sensitivity and specificity for VSIV as the previously published assay. Our results highlight the challenges the large genetic variability of VSV pose for virus detection by mRRT-PCR and show the importance of frequent re-evaluation and validation of diagnostic assays for VSV to ensure high sensitivity and specificity.

Using a Polymerase Chain Reaction as a Complementary Test to Improve the Detection of Dermatophyte Fungus in Nails

2014 ◽  
Vol 104 (3) ◽  
pp. 233-237 ◽  
Author(s):  
María José Iglesias Sánchez ◽  
Ana María Pérez Pico ◽  
Félix Marcos Tejedor ◽  
María Jesús Iglesias Sánchez ◽  
Raquel Mayordomo Acevedo
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Classical Method ◽  
Clinical Manifestations ◽  
Culture Method ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Traditional Procedure ◽  
Polymerase Chain

Background Dermatomycoses are a group of pathologic abnormalities frequently seen in clinical practice, and their prevalence has increased in recent decades. Diagnostic confirmation of mycotic infection in nails is essential because there are several pathologic conditions with similar clinical manifestations. The classical method for confirming the presence of fungus in nail is microbiological culture and the identification of morphological structures by microscopy. Methods We devised a nested polymerase chain reaction (PCR) that amplifies specific DNA sequences of dermatophyte fungus that is notably faster than the 3 to 4 weeks that the traditional procedure takes. We compared this new technique and the conventional plate culture method in 225 nail samples. The results were subjected to statistical analysis. Results We found concordance in 78.2% of the samples analyzed by the two methods and increased sensitivity when simultaneously using the two methods to analyze clinical samples. Now we can confirm the presence of dermatophyte fungus in most of the positive samples in just 24 hours, and we have to wait for the result of culture only in negative PCR cases. Conclusions Although this PCR cannot, at present, substitute for the traditional culture method in the detection of dermatophyte infection of the nails, it can be used as a complementary technique because its main advantage lies in the significant reduction of time used for diagnosis, in addition to higher sensitivity.

Sign in / Sign up

Export Citation Format

Share Document