Nested PCR method for rapid and sensitive detection of Vibrio vulnificus in fish, sediments, and water.

Abstract We developed new ITD detection algorithm (ITDetect) based on whole exome sequencing (WES) data for FMS-like tyrosine kinase internal tandem duplication (FLT3-ITD). ITDetect is based on BWA and is specified for ITD detection including FLT3-ITD. We validated and compared result of ITDetect with other ITD detecting algorithms using nested polymerase chain reaction (PCR) method. Nested PCR uses two types of primer specified for FLT3-ITD detection. In 81 acute myeloid leukemia patients with WES data, FLT3-ITD was positive in 11 patients (13%) when called with ITDetect, all of whom were validated with nested PCR. Meanwhile FLT3-ITD was positive only in 7/81 patients by conventional PCR. The concordance rate of ITDetect and nested PCR was 95% (77/81). ITDetect showed better ITD detection performance when compared with previously reported ITD callers. In large AML cohort (n=213), patients who were positive for FLT3-ITD with nested-PCR but not with conventional PCR had shorter survival outcomes than patients who were negative for FLT3-ITD with nested PCR, suggesting clinical significance of sensitive FLT3-ITD detection. In conclusion, we developed more sensitive detection methods for FLT3-ITD based on WES data that is clinically meaningful. Utilization of more sensitive detection method than conventional PCR in clinic should be considered. Figure FLT3-ITD detection procedure. Figure. FLT3-ITD detection procedure. Figure Performance of various NGS ITD detectors. Figure. Performance of various NGS ITD detectors. Figure Survival comparison between patients positive for FLT3-ITD by nested PCR method but negative by conventional PCR method and those negative by both methods. Figure. Survival comparison between patients positive for FLT3-ITD by nested PCR method but negative by conventional PCR method and those negative by both methods. Disclosures No relevant conflicts of interest to declare.

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Rapid concentration and sensitive detection of hookworm ova from wastewater matrices using a real-time PCR method

Experimental Parasitology ◽

10.1016/j.exppara.2015.08.009 ◽

2015 ◽

Vol 159 ◽

pp. 5-12 ◽

Cited By ~ 17

Author(s):

P. Gyawali ◽

J.P.S. Sidhu ◽

W. Ahmed ◽

P. Jagals ◽

S. Toze

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Sensitive Detection ◽

Pcr Method ◽

Hookworm Ova

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Development of a one step real-time RT-PCR method for sensitive detection of human astrovirus

Journal of Virological Methods ◽

10.1016/j.jviromet.2005.10.012 ◽

2006 ◽

Vol 133 (1) ◽

pp. 14-19 ◽

Cited By ~ 15

Author(s):

Enrique Royuela ◽

Ana Negredo ◽

Alicia Sánchez-Fauquier

Keyword(s):

Real Time ◽

Sensitive Detection ◽

Rt Pcr ◽

One Step ◽

Human Astrovirus ◽

Pcr Method

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Efficient nested-PCR-based method development for detection and genotype identification of Acanthamoeba from a small volume of aquatic environmental sample

Scientific Reports ◽

10.1038/s41598-021-00968-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tsui-Kang Hsu ◽

Jung-Sheng Chen ◽

Hsin-Chi Tsai ◽

Chi-Wei Tao ◽

Yu-Yin Yang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Nested Pcr ◽

In Silico ◽

Environmental Samples ◽

Method Development ◽

Small Volume ◽

Detection Efficiency ◽

Genotyping Method ◽

Pcr Method

AbstractAcanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

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Direct Identification of Vibrio vulnificus in Clinical Specimens by Nested PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.36.10.2887-2892.1998 ◽

1998 ◽

Vol 36 (10) ◽

pp. 2887-2892 ◽

Cited By ~ 40

Author(s):

Shee Eun Lee ◽

Soo Young Kim ◽

Sei Jong Kim ◽

Hyun Soo Kim ◽

Jong Hee Shin ◽

...

Keyword(s):

Dna Extraction ◽

Nested Pcr ◽

Extraction Method ◽

Vibrio Vulnificus ◽

High Yield ◽

Chromosomal Dna ◽

Direct Identification ◽

Clinical Specimens ◽

Dna Extraction Method ◽

Dna Fragment

This study was performed to establish optimal nested PCR conditions and a high-yield DNA extraction method for the direct identification ofVibrio vulnificus in clinical specimens. We designed two sets of primers targeting the V. vulnificushemolysin/cytolysin gene. The target of the first primer set (P1-P2; sense, 5′-GAC-TAT-CGC-ATC-AAC-AAC-CG-3′, and antisense, 5′-AGG-TAG-CGA-GTA-TTA-CTG-CC-3′, respectively) is a 704-bp DNA fragment. The second set (P3-P4; sense, 5′-GCT-ATT-TCA-CCG-CCG-CTC-AC-3′, and antisense, 5′-CCG-CAG-AGC-CGT-AAA-CCG-AA-3′, respectively) amplifies an internal 222-bp DNA fragment. We developed a direct DNA extraction method that involved boiling the specimen pellet in a 1 mM EDTA–0.5% Triton X-100 solution. The new DNA extraction method was more sensitive and reproducible than other conventional methods. The DNA extraction method guaranteed sensitivity as well, even when V. vulnificus cells were mixed with other bacteria such asEscherichia coli or Staphylococcus aureus. The nested PCR method could detect as little as 1 fg of chromosomal DNA and single CFU of V. vulnificus. We applied the nested PCR protocol to a total of 39 serum specimens and bulla aspirates from septicemic patients. Seventeen (94.4%) of the 18V. vulnificus culture-positive specimens were positive by the nested PCR. Eight (42.1%) of the 19 culture-negative samples gave positive nested PCR results.

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Dual-target one-step nested PCR for sensitive detection of SARS-CoV-2 nucleic acids

Preparative Biochemistry & Biotechnology ◽

10.1080/10826068.2021.1964084 ◽

2021 ◽

pp. 1-7

Author(s):

Qijie Li ◽

Yiqing Xia ◽

Dunshui Liao ◽

Hu Nie ◽

Ming Zhang ◽

...

Keyword(s):

Nucleic Acids ◽

Nested Pcr ◽

Sensitive Detection ◽

One Step ◽

Dual Target

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Estimating Giardia cyst viability using RT-PCR

Water Science & Technology Water Supply ◽

10.2166/ws.2002.0092 ◽

2002 ◽

Vol 2 (3) ◽

pp. 107-115 ◽

Cited By ~ 1

Author(s):

M. Maux ◽

I. Bertrand ◽

C. Gantzer ◽

J. Schwartzbrod

Keyword(s):

Nested Pcr ◽

Target Genes ◽

Molecular Techniques ◽

Giardia Cyst ◽

Health Issues ◽

Structural Protein ◽

Gene Encoding ◽

Response To Stress ◽

Pcr Method ◽

Giardia Cysts

The aim of this work was to evaluate the use of molecular techniques for the detection of viable Giardia cysts in the environment to assess public health issues. Three target genes were selected: the heat shock protein gene, HSP70, which is expressed in response to stress; the giardin gene, which encodes a structural protein; and, alcohol dehydrogenase E (ADHE), a novel gene encoding an enzyme involved in the metabolism of energy. We tested the efficiency of five protocols for the extraction of either genomic DNA or total RNA from Giardia cysts: two of these protocols were previously cited in the literature and three consisted of commercial DNA extraction kits. The brands of enzyme were determined according to the primers chosen and the amplification conditions were optimised: 2.5 mM MgCl2, 0.5 mM primers and 60°C for annealing temperature. A semi-nested PCR method and an RT semi-nested PCR procedure were developed to detect mRNA from these three genes and to estimate the viability of Giardia cysts.

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