scholarly journals Multiplex PCR for detection of MCR genes in clinical fecal samples

2021 ◽  
Vol 269 ◽  
pp. 01019
Author(s):  
Qiumei Xiang ◽  
Shuanglan Hu ◽  
Yuebin Ke ◽  
Shuangfang Hu
Keyword(s):  
Clinical Laboratory ◽  
Sequence Data ◽  
Rapid Identification ◽  
Whole Genome Sequence ◽  
Epidemiological Surveillance ◽  
Universal Primers ◽  
Colistin Resistance ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Higher Sensitivity

Plasmid-mediated colistin-resistance genes have been reported worldwide in recent years. A multiplex polymerase chain reaction (Multi-PCR) protocol was developed to detect transferable colistinresistance genes (mcr-1 to mcr-6) in Enterobacteria for clinical laboratory purposes.The authors first designed six new primer pairs to amplify mcr-1 to mcr-6 gene products to achieve stepwise separation of amplicons between 87 to 216 bp,then divided these primers into two subgroups with the assistance of a pair of universal primers for the detection of currently described mcr genes and their variants in Enterobacteria. The protocol was validated by testing 29 clinical isolates of Escherichia coli of human origin, each well characterised and prospectively validated. The Multi-PCR assay showed full concordance with whole-genome sequence data and displayed higher sensitivity and 100% specificity. The assay could detect all variants of the various mcr alleles described. It was able to detect mcr-3 and mcr-4 as singletons or in combination. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance in limited resources conditions, and this method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance.

scholarly journals Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes

2018 ◽  
Vol 23 (6) ◽  
Author(s):  
Ana Rita Rebelo ◽  
Valeria Bortolaia ◽  
Jette S Kjeldgaard ◽  
Susanne K Pedersen ◽  
Pimlapas Leekitcharoenphon ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Sequence Data ◽  
Resistance Mechanisms ◽  
Rapid Identification ◽  
Whole Genome Sequence ◽  
Animal Origin ◽  
Colistin Resistance ◽  
The Past ◽  
Multiple Detection ◽  
Polymerase Chain

Background and aim Plasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.6**, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.

scholarly journals A PCR-based Technique for Identification of Fusicoccum sp. from Pistachio and Various Other Hosts in California

Plant Disease ◽  
2002 ◽  
Vol 86 (5) ◽  
pp. 515-520 ◽  
Author(s):  
Zhonghua Ma ◽  
Themis J. Michailides
Keyword(s):  
Host Plants ◽  
Extraction Procedure ◽  
Its Region ◽  
Pcr Primers ◽  
Rapid Identification ◽  
Fungal Species ◽  
Pcr Assay ◽  
Shoot Blight ◽  
Dna Fragment ◽  
Polymerase Chain

Botryosphaeria panicle and shoot blight of pistachio, caused by Fusicoccum sp. is a destructive disease in California. In this study, a pair of group-specific polymerase chain reaction (PCR) primers BDI and BDII, was developed for identification of Fusicoccum sp. from pistachio and other hosts in California based on the sequences of the rDNA internal transcribed spacer (ITS) region. The primers amplified a 356-bp DNA fragment for all 73 tested isolates of Fusicoccum sp. collected from pistachio and other hosts throughout California in different years, but not for the other 33 fungal species isolated from pistachio and the eight isolates of Fusicoccum sp. obtained from pistachio trees in Greece. The PCR assay using this pair of primers was sensitive enough to detect 5 pg of genomic DNA of Fusicoccum sp. A simple DNA extraction procedure was developed that led to the rapid identification of Fusicoccum sp. from pistachio and other host plants in California.

scholarly journals Molecular Assay Validation Using Genomic Sequence Databases

2016 ◽  
Vol 54 (12) ◽  
pp. 2854-2856 ◽  
Author(s):  
John P. Dekker
Keyword(s):  
Clinical Microbiology ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Whole Genome Sequence ◽  
Clinical Microbiology Laboratory ◽  
Microbiology Laboratory ◽  
Molecular Assay ◽  
Pcr Assay ◽  
Content Type ◽  
Sequence Databases

Whole-genome sequence databases offer newin silicoapproaches for designing and validating PCR assays in the clinical microbiology laboratory. An article in this issue of theJournal of Clinical Microbiology(M. J. Jansen van Rensburg, C. Swift, A. J. Cody, C. Jenkins, and M. C. J. Maiden, J Clin Microbiol, 54:2882–2890, 2016,http://dx.doi.org/10.1128/JCM.01522-16) demonstrates the use of publicly available genomic sequence data to evaluate a PCR assay for distinguishingCampylobacterspecies.

scholarly journals Rapid identification of melioidosis agent by an insulated isothermal PCR on a field–deployable device

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9238
Author(s):  
Kek Heng Chua ◽  
E. Wei Tan ◽  
Hwa Chia Chai ◽  
SD Puthucheary ◽  
Ping Chin Lee ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Clinical Laboratory ◽  
Rapid Identification ◽  
Chain Reaction ◽  
Serious Illness ◽  
Lower Detection Limit ◽  
Lower Detection ◽  
Polymerase Chain ◽  
Intracellular Motility ◽  
Insulated Isothermal Pcr

Background Burkholderia pseudomallei causes melioidosis, a serious illness that can be fatal if untreated or misdiagnosed. Culture from clinical specimens remains the gold standard but has low diagnostic sensitivity. Method In this study, we developed a rapid, sensitive and specific insulated isothermal Polymerase Chain Reaction (iiPCR) targeting bimA gene (Burkholderia Intracellular Motility A; BPSS1492) for the identification of B. pseudomallei. A pair of novel primers: BimA(F) and BimA(R) together with a probe were designed and 121 clinical B. pseudomallei strains obtained from numerous clinical sources and 10 ATCC non-targeted strains were tested with iiPCR and qPCR in parallel. Results All 121 B. pseudomallei isolates were positive for qPCR while 118 isolates were positive for iiPCR, demonstrating satisfactory agreement (97.71%; 95% CI [93.45–99.53%]; k = 0.87). Sensitivity of the bimA iiPCR/POCKIT assay was 97.52% with the lower detection limit of 14 ng/µL of B. pseudomallei DNA. The developed iiPCR assay did not cross-react with 10 types of non-targeted strains, indicating good specificity. Conclusion This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen.

Automated DNA sequencing methods involving polymerase chain reaction.

1989 ◽  
Vol 35 (11) ◽  
pp. 2196-2201 ◽  
Author(s):  
L J McBride ◽  
S M Koepf ◽  
R A Gibbs ◽  
W Salser ◽  
P E Mayrand ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequencing ◽  
Dna Sequence ◽  
Sequence Data ◽  
Identification Accuracy ◽  
Universal Primers ◽  
Chain Reaction ◽  
Universal Primer ◽  
Polymerase Chain ◽  
Primer Sequence

Abstract Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.

Development of a fully automated PCR assay for the detection of Pneumocystis jirovecii using the GENECUBE system

2018 ◽  
Vol 57 (7) ◽  
pp. 841-847 ◽  
Author(s):  
Yasufumi Matsumura ◽  
Yasuhiro Tsuchido ◽  
Masaki Yamamoto ◽  
Satoshi Nakano ◽  
Miki Nagao
Keyword(s):  
Real Time ◽  
Clinical Laboratory ◽  
Pneumocystis Pneumonia ◽  
Pneumocystis Jirovecii ◽  
Chain Reaction ◽  
Laboratory Practice ◽  
Pcr Assay ◽  
Promising Tool ◽  
Polymerase Chain ◽  
Routine Clinical Laboratory

AbstractWe developed a fully automated polymerase chain reaction (PCR) assay for the detection of Pneumocystis jirovecii using the GENECUBE system. This assay was evaluated against an in-house real-time PCR assay using 82 bronchoalveolar lavage and 139 sputum samples from 221 immunocompromised patients who were suspected of having Pneumocystis pneumonia (PCP). After loading the maximum of eight samples into the GENECUBE system, the results were obtained within approximately 60 minutes. The overall positivity rate of both assays was 35%, and the concordance rate was 89% (kappa, 0.76). Based on the clinical diagnosis of 39 PCP and 105 non-PCP patients, the GENECUBE and real-time assays had sensitivities of 92.3% and 94.9% and specificities of 85.7% and 85.7%, respectively. This automated and rapid assay is a promising tool for the detection of P. jirovecii in routine clinical laboratory practice.

scholarly journals ConFindr: rapid detection of intraspecies and cross-species contamination in bacterial whole-genome sequence data

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6995 ◽  
Author(s):  
Andrew J. Low ◽  
Adam G. Koziol ◽  
Paul A. Manninger ◽  
Burton Blais ◽  
Catherine D. Carrillo
Keyword(s):  
Sequence Data ◽  
Single Copy ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Computational Pipeline ◽  
Ribosomal Protein Genes ◽  
Multiple Alleles ◽  
Quality Issue ◽  
Unrelated Strain ◽  
Higher Sensitivity

Whole-genome sequencing (WGS) of bacterial pathogens is currently widely used to support public-health investigations. The ability to assess WGS data quality is critical to underpin the reliability of downstream analyses. Sequence contamination is a quality issue that could potentially impact WGS-based findings; however, existing tools do not readily identify contamination from closely-related organisms. To address this gap, we have developed a computational pipeline, ConFindr, for detection of intraspecies contamination. ConFindr determines the presence of contaminating sequences based on the identification of multiple alleles of core, single-copy, ribosomal-protein genes in raw sequencing reads. The performance of this tool was assessed using simulated and lab-generated Illumina short-read WGS data with varying levels of contamination (0–20% of reads) and varying genetic distance between the designated target and contaminant strains. Intraspecies and cross-species contamination was reliably detected in datasets containing 5% or more reads from a second, unrelated strain. ConFindr detected intraspecies contamination with higher sensitivity than existing tools, while also being able to automatically detect cross-species contamination with similar sensitivity. The implementation of ConFindr in quality-control pipelines will help to improve the reliability of WGS databases as well as the accuracy of downstream analyses. ConFindr is written in Python, and is freely available under the MIT License at github.com/OLC-Bioinformatics/ConFindr.

scholarly journals Rapid and Sensitive Detection of Phytophthora sojae in Soil and Infected Soybeans by Species-Specific Polymerase Chain Reaction Assays

2006 ◽  
Vol 96 (12) ◽  
pp. 1315-1321 ◽  
Author(s):  
Yuanchao Wang ◽  
Wenli Zhang ◽  
Ying Wang ◽  
Xiaobo Zheng
Keyword(s):  
Polymerase Chain Reaction ◽  
Screening Method ◽  
Phytophthora Species ◽  
Phytophthora Sojae ◽  
Detection Sensitivity ◽  
Universal Primers ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Soybean Tissue

Root and stem rot caused by Phytophthora sojae is one of the most destructive diseases of soybean (Glycine max) worldwide. P. sojae can survive as oospores in soil for many years. In order to develop a rapid and accurate method for the specific detection of P. sojae in soil, the internal transcribed spacer (ITS) regions of eight P. sojae isolates were amplified using polymerase chain reaction (PCR) with the universal primers DC6 and ITS4. The sequences of PCR products were aligned with published sequences of 50 other Phytophthora species, and a region specific to P. sojae was used to design the specific PCR primers, PS1 and PS2. More than 245 isolates representing 25 species of Phytophthora and at least 35 other species of pathogens were used to test the specificity of the primers. PCR amplification with PS primers resulted in the amplification of a product of approximately 330 bp, exclusively from isolates of P. sojae. Tests with P. sojae genomic DNA determined that the sensitivity of the PS primer set is approximately 1 fg. This PCR assay, combined with a simple soil screening method developed in this work, allowed the detection of P. sojae from soil within 6 h, with a detection sensitivity of two oospores in 20 g of soil. PCR with the PS primers could also be used to detect P. sojae from diseased soybean tissue and residues. Real-time fluorescent quantitative PCR assays were also developed to detect the pathogen directly in soil samples. The PS primer-based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in soil and infected soybean tissue.

Development and Evaluation of 16S rDNA Microarray for Detecting Bacterial Pathogens in Cerebrospinal Fluid

2005 ◽  
Vol 230 (8) ◽  
pp. 587-591 ◽  
Author(s):  
Yi Liu ◽  
Jin-Xiang Han ◽  
Hai-Yan Huang ◽  
Bo Zhu
Keyword(s):  
Cerebrospinal Fluid ◽  
16S Rdna ◽  
Culture Media ◽  
Sequence Data ◽  
Rapid Identification ◽  
Universal Primers ◽  
16S Rdna Sequence ◽  
Rdna Sequence Data ◽  
Identification Of Bacteria ◽  
Culture Positive

The rapid identification of bacteria in cerebrospinal fluid (CSF) is very Important for patient management and antimicrobial therapies. We developed a 16S DNA microarray–based method that targets 16S rDNA and can directly detect bacteria from CSF without cultivation. Universal primers and specific probes were designed from the 16S rDNA sequence data retrieved directly from the GenBank database. The specificity of the assay is obtained through a combination of microarray hybridization and enzymatic labeling of the constructed specific probes. Cultivation-dependent assays were used as reference methods in the development and evaluation of the method. With the exception of Mycobacterium tuberculosis and Proteus mirabilis, forty-five positive blood culture media were successfully differentiated. When this procedure was applied directly to 100 CSF specimens, 29 specimens from 16 patients were positive by bacterial culture and 3 culture-positive CSF specimens produced no hybridized signals. The remaining 26 specimens were correctly identified, including one with mixed infection. The accuracy, sensitivity, and specificity of the assay can be increased further by designing more oligonucleotides for the microarray. This method is versatile and makes it possible to detect more bacteria in a single assay and discriminate different bacterial genera.

A Real-Time PCR Assay for Rapid Identification of Tuta absoluta (Lepidoptera: Gelechiidae)

2020 ◽  
Vol 113 (3) ◽  
pp. 1479-1485
Author(s):  
Frida A Zink ◽  
Luke R Tembrock ◽  
Alicia E Timm ◽  
Todd M Gilligan
Keyword(s):  
Real Time ◽  
The United States ◽  
Rapid Identification ◽  
Tuta Absoluta ◽  
Morphological Identification ◽  
Pcr Assay ◽  
Tomato Crop ◽  
The North ◽  
Polymerase Chain ◽  
Imminent Threat

Abstract The tomato leafminer, Tuta absoluta (Meyrick), is a highly destructive pest of tomatoes, causing damage to leaves, stalks, buds, and fruits. Native to South America, T. absoluta is now found throughout Europe, South Asia, Africa, parts of Central America, and the Caribbean. Adults are small, with a wingspan of approximately one cm and lack distinctive markings, making morphological identification difficult. Larvae are also difficult to identify and resemble those of many other gelechiids. Due to the extensive time spent and expertise required for morphological identification, and the imminent threat to the North American tomato crop, we have developed a rapid molecular test for discriminating individual specimens of T. absoluta using a probe-based real-time polymerase chain reaction (PCR) assay. The assay is able to quickly distinguish T. absoluta from similar-sized moth specimens that are attracted to T. absoluta pheromone lures in the United States and is also able to identify larvae of T. absoluta. Decreased identification time for this critical pest will lead to more rapid identification at ports of entry and allow for more efficient trap screening for domestic monitoring programs.

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