scholarly journals Comparison of Two DNA Extraction Methods and Two PCRs for Detection of Echinococcus multilocularis in the Stool Samples of Naturally Infected Red Foxes

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2381
Author(s):  
Katarzyna Skrzypek ◽  
Jacek Karamon ◽  
Małgorzata Samorek-Pieróg ◽  
Joanna Dąbrowska ◽  
Maciej Kochanowski ◽  
...  
Keyword(s):  
Positive Result ◽  
Multiplex Pcr ◽  
Dna Extraction ◽  
Echinococcus Multilocularis ◽  
Nested Pcr ◽  
Genetic Material ◽  
Extraction Methods ◽  
Red Foxes ◽  
Stool Samples ◽  
Positive Results

(1) Background: Due to the increasing distribution of Echinococcus multilocularis infections in final hosts, epidemiological investigations are important for recognizing the spreading pattern of this parasite and also to estimate risk infection for humans. (2) Methods: Investigations were conducted with two commercial kits dedicated for DNA extraction from feces: ZR Fecal DNA Mini Prep (Zymo Research, Freiburg, Germany) and QIAamp FAST DNA Stool Mini Kit (Qiagen, Hilden, Germany) (marked as Z and Q), together with two common PCR protocols (nested PCR and multiplex PCR). The goal was to compare their efficiency in detecting the genetic material of E. multilocularis in the samples of feces. Stool samples from red foxes were collected in a highly endemic area in Poland. Sedimentation and counting technique (SCT) was used as a reference method. (3) Results: From 48 samples, 35 were positive in SCT. Further investigations showed that 40.0% of samples (from those with SCT positive result) after Z-DNA extraction and 45.7% after Q-DNA extraction gave positive results in nested PCR. In multiplex PCR, positive results were obtained in 54.3% of samples after Z isolation and 48.6% of samples after Q. Additionally, one sample that resulted in being negative in SCT gave a positive result in PCR. The number of worms detected in the intestines had no influence on PCR results. (4) Conclusions: Both of the extraction methods showed similar efficiency in DNA isolation and dealing with inhibitors; however, they showed relatively low sensitivity. This was probably caused by degradation of genetic material in the field-collected samples.

Detection of Echinococcus multilocularis in faeces by nested PCR with the use of diluted DNA samples

2014 ◽  
Vol 17 (1) ◽  
pp. 79-83 ◽  
Author(s):  
J. Karamon
Keyword(s):  
Echinococcus Multilocularis ◽  
Nested Pcr ◽  
Dna Isolation ◽  
False Negative ◽  
Standard Samples ◽  
Red Foxes ◽  
Negative Results ◽  
False Negative Results ◽  
Faecal Samples ◽  
Positive Results

AbstractThe aim of this study was to choose the optimal variant of PCR examination of faeces to detect Echinococcus multilocularis infection which would allow to reduce the influence of different inhibitors in faeces. The investigation was carried out by comparison of 3 different methods of DNA isolation from faeces and different DNA dilutions used in PCR. Thirty five intestines of red foxes were used. Small intestines were examined by the sedimentation and counting technique (SCT). Faeces were collected from the rectum for PCR and flotation. DNA were isolated with the use of 3 different methods. Two methods were dedicated for faeces: method 1 (M1) - for larger samples and method 2 (M2) - for standard samples. The third method, method 3 (M3), was not dedicated for faeces. DNA samples were tested by nested PCR in 6 variants: not diluted (1/1) and 5 diluted (1/2.5, 1/5, 1/10. 1/20, 1/40). E. multilocularis was found by SCT in 18 from 35 (51.4%) intestines. Taenia-type eggs were detected only in 20.0% of faecal samples. In PCR the highest number of positive results (45.7%) were obtained during examination of DNA isolated by M1 method, and then 40.0% and 34.3%, respectively, for M2 and M3. In some samples positive results in PCR were obtained only in diluted DNA. For example, 8 from 12 positive samples isolated by M3 method gave the PCR negative results in non-diluted DNA and positive only after dilution 1:2.5, 1:10 or 1:20. Also 3 samples isolated by methods dedicated for stool gave positive results only after DNA dilution. The investigation has revealed that in copro-PCR for detection of E. multilocularis infection additional using of diluted DNA (besides non diluted) can avoid false negative results causing by PCR inhibition. In the best method of DNA isolation (M1), the use of non diluted DNA sample together with diluted in proportion 1:10 seems to be optimal.

Simple modification to improve reliability of copro-DNA examinations for diagnosing Echinococcus multilocularis infections in red foxes

2020 ◽  
Vol 94 ◽  
Author(s):  
T. Irie ◽  
T. Ito ◽  
H. Kouguchi ◽  
K. Uraguchi
Keyword(s):  
Dna Extraction ◽  
Echinococcus Multilocularis ◽  
Epidemiological Studies ◽  
Sequencing Analysis ◽  
Red Foxes ◽  
Simple Modification ◽  
Pcr Inhibitors ◽  
Examination Technique ◽  
Modified Method ◽  
Infection Pressure

Abstract Epidemiological studies of Echinococcus multilocularis infections in definitive hosts require a reliable and economic diagnostic method. In this study, the current copro-DNA examination technique was modified by increasing the faecal amounts tested and adding a step to neutralize the faeces before DNA extraction. Reliability of the modified method was evaluated using rectal faecal samples from red foxes and comparing them with intestinal worms detected using the sedimentation and counting technique (SCT) following necropsy. The modified copro-DNA examination method demonstrated 93.9% sensitivity (138/147) on the SCT. Its detectability increased depending on the worm burden, and the sensitivity was 100% in cases harbouring over 1000 worms. From 111 SCT-negative cases, six (5.4%) were copro-DNA-positive, and all were confirmed as E. multilocularis via sequencing analysis. Five of the remaining 105 SCT-negative cases (4.8%) retained polymerase chain reaction (PCR) inhibitors in the extracted solution, suggesting that approximately 5% of the red fox faeces retained these inhibitors after treatment with the present copro-DNA extraction method. Although further evaluation is needed for faeces deposited in the wild, the present copro-DNA examination technique will help monitor the E. multilocularis prevalence in definitive hosts. When used for detailed evaluations of endemicity (e.g. changes in infection pressure or spread in non-endemic areas), the absence of PCR inhibitors should be confirmed, and multiple trials on faecal subsamples are recommended.

scholarly journals P851 Effects of conservation time on the diversity and relative abundance of intestinal microbiome in samples of faecal matters

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S655-S655
Author(s):  
M León F ◽  
C A Nieto ◽  
Z Corredor ◽  
C Flórez-Sarmiento ◽  
V Parra-Izquierdo ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Relative Abundance ◽  
High Reliability ◽  
Genetic Material ◽  
Variable Region ◽  
Important Variable ◽  
Intestinal Microbiome ◽  
Three Samples ◽  
Stool Samples ◽  
Changes Over Time

Abstract Background Given the complexity and diversity of the intestinal microbiome, there is a technical challenge of finding the best way to study the factors that affect the quality of the sample to obtain results in a precise an complete way. Objective To establish the effect of storage time under constant cryopreservation conditions (−20°C) in stool samples for the study of the gastrointestinal microbiome Methods A sample of stool was distributed in 3 fractions (1, 2 and 3). Time 0 without cryopreservation and immediate DNA extraction (1). The samples of 15 (2) and 30 (3) days were cryopreserved at −20°C before the extraction of the genetic material. After ultracentrifugation at 4 degrees Celsius, the precipitate was subjected to enzymatic and mechanical lysis to obtain the total DNA. The DNA quality was evaluated techniques to ensure the quality and concentration of genetic material. Once the DNA of the three samples was obtained, they were subjected to the latest generation of a variable region of the 16S gene, using MiSeq technology (Illuminates). Data in relative abundances and frequencies were recorded. Project supported by the Administrative Department of Science, Technology and Innovation-Colciencias Health Call 2017, Code 130877757442 Results About 140 thousand readings were obtained for samples 1 (day 0) and 2 (day 15) and 160 thousand for sample 3 (day 30), with a reading quality Q20% between 97 and 98 and Q30% around 94, indicating a high-reliability value for each of the samples. Regarding the classification from the Operating Taxonomic Unit (OTU), 119, 99 and 106 were reported for samples 1, 2 and 3, respectively. The data obtained revealed changes over time at the level of the main edges reported for normal microbiome (Bacteroidetes, Firmicutes, Actinobacteria) between 1 and 2 vs. sample 3. For Firmicutes and Actinobacteria, decreases in abundance of 34% were observed (samples 1 and 2) at 20% for Firmicutes in sample 3 and from 4% (samples 1 and 2) to 0.8% (sample 3) for Actinobacteria. On the contrary, Bacteroidetes presented an increase in its abundance from 58% (Samples 1 and 2) to 76% (sample 3). The Proteobacterium edge did not show significant changes in its abundance Conclusion It was possible to demonstrate that the cryopreservation time before DNA extraction is an important variable that influences the percentage and integrity of the microbiota from faecal matter. Confirming that the maximum reliable shelf life at −20° in stool samples is up to 15 days, suggesting that for longer storage the temperature decrease up to −80° should be taken into account to maintain stability in the results of relative abundance and bacterial diversity

Use of sodium hydroxide DNA extraction methods for nested PCR detection of Bretziella fagacearum in the sapwood of oak species in Minnesota

2021 ◽  
Author(s):  
Melanie Joy Moore ◽  
Jennifer Juzwik ◽  
Olga Saiapina ◽  
Snober Ahmed ◽  
Anna Yang ◽  
...  
Keyword(s):  
Sodium Hydroxide ◽  
Dna Extraction ◽  
Nested Pcr ◽  
Pathogen Detection ◽  
Abiotic Factors ◽  
Laboratory Diagnosis ◽  
Extraction Methods ◽  
White Oak ◽  
Detection Rates ◽  
Quercus Species

Oak wilt caused by Bretziella fagacearum is an important disease of Quercus species, but its diagnosis may be confused with damage resulting from other diseases, insects, or abiotic factors. Laboratory diagnosis is important in such situations and when disease control action is desired. Polymerase chain reaction (PCR) tests can provide accurate lab diagnosis within two days. Two variations of a simple DNA extraction protocol using sodium hydroxide (NaOH) were compared to that of the proprietary protocol of a commercially available kit (CK) for nested PCR to detect the pathogen in oak sapwood. High frequencies of pathogen detection (98 to 100% of 48 branch segments assayed) were found for northern pin oak using the two NaOH-based and the CK methods. Detection rates were similar but lower for bur oak (ranged from 58 to 79%) and white oak (ranged from 54 to 71%) regardless of DNA extraction method. Using our alternative DNA extraction protocols may reduce total time and cost of B. fagacearum detection in PCR-based diagnosis and other downstream applications.

scholarly journals Multicenter Comparative Study of Six Cryptosporidium parvum DNA Extraction Protocols Including Mechanical Pretreatment from Stool Samples

2020 ◽  
Vol 8 (9) ◽  
pp. 1450
Author(s):  
Nicolas Valeix ◽  
Damien Costa ◽  
Louise Basmaciyan ◽  
Stéphane Valot ◽  
Anne Vincent ◽  
...  
Keyword(s):  
Comparative Study ◽  
Real Time ◽  
Dna Extraction ◽  
Cryptosporidium Parvum ◽  
Real Time Pcr ◽  
Sample Pretreatment ◽  
Dna Amplification ◽  
Extraction Methods ◽  
Mechanical Pretreatment ◽  
Stool Samples

Background: Nowadays, many commercial kits allow the detection of Cryptosporidium sp. in stool samples after deoxyribonucleic acid (DNA) extraction. Protocols of stool pretreatment have been proposed to optimize oocysts’ DNA extraction. Among them, mechanical grinding was reported to improve the performance of Cryptosporidium oocysts’ DNA extraction. Methods: A multicenter comparative study was conducted within the framework of the French National Reference Center-Expert Laboratory for Cryptosporidiosis. Six extraction systems (i.e., manual or automated) associated with various mechanical pretreatment protocols, were compared for the Cryptosporidium parvum oocyst’ DNA extraction, before amplification using the same real-time PCR method targeting. Results: The sensitivity of real-time PCR assay was unequally impacted by the pretreatment/extraction protocol. We observed significant differences for the lowest concentrations of C. parvum oocysts (i.e., 0–94.4% and 33.3–100% respectively for 10 and 50 oocysts/mL). All in all, the protocol using Quick DNA Fecal/Soil Microbe-Miniprep® manual kit showed the best performances. In addition, optimal performances of mechanical pretreatment were obtained by combining a grinding duration of 60 s with a speed of 4 m/s using Fastprep24® with Lysing Matrix E®. Conclusions: Sample pretreatment, as well as the extraction method, needs to be properly adapted to improve the diagnostic performances of the C. parvum DNA amplification methods.

scholarly journals Comparison of Three Mycobacterial DNA Extraction Methods from Extrapulmonary Samples for PCR Assay

2013 ◽  
Vol 6 (1) ◽  
pp. 9-11
Author(s):  
Khandaker Shadia ◽  
Shaheda Anwar ◽  
Sayera Banu ◽  
Ahmed Abu Saleh ◽  
Md Ruhul Amin Miah
Keyword(s):  
Dna Extraction ◽  
Extrapulmonary Tuberculosis ◽  
Extraction Methods ◽  
Molecular Diagnostic ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Lysis Buffer ◽  
Dna Extraction Methods ◽  
Positive Results

Sensitivity of the molecular diagnostic tests of extrapulmonary tuberculosis largely depends upon the efficiency of DNA extraction methods. The objective of our study was to compare three methods of extracting DNA of Mycobacterium tuberculosis for testing by polymerase chain reaction. All three methods; heating, heating with sonication and addition of lysis buffer with heating and sonication were implicated on 20 extrapulmonary samples. PCR positivity was 2 (10%), 4 (20%) and 7 (35%) in the samples extracted by heating, heat+sonication and heat+sonication+lysis buffer method respectively. Of the extraction methods evaluated, maximum PCR positive results were achieved by combined heat, sonication and lysis buffer method which can be applied in routine clinical practice. DOI: http://dx.doi.org/10.3329/imcj.v6i1.14711 Ibrahim Med. Coll. J. 2012; 6(1): 9-11

scholarly journals Comparison of techniques for DNA extraction and agarose gel staining of DNA fragments using samples of Cryptosporidium

2013 ◽  
Vol 58 (No. 10) ◽  
pp. 535-542 ◽  
Author(s):  
MCM Couto ◽  
AP Sudre ◽  
MF Lima ◽  
TCB Bomfim
Keyword(s):  
Dna Extraction ◽  
Ethidium Bromide ◽  
Ribosomal Rna ◽  
Nested Pcr ◽  
Extraction Methods ◽  
Small Subunit ◽  
Molecular Techniques ◽  
Comparison Of Techniques ◽  
Low Toxicity ◽  
Dna Extraction Methods

Differentiating between the Cryptosporidium species and their subtypes using only microscopy is impossible. Therefore, molecular tools are indispensable for accurate species and subtype diagnosis. However, if these tools are to be used correctly and accurately, the techniques used must be standardised. In the present study, two molecular techniques for diagnosing Cryptosporidium infection in cows were compared to determine the optimal methods. For each technique, we tested two DNA extraction methods, several annealing temperatures for nested PCR reactions targeting the 18S, SSU rRNA (small subunit ribosomal RNA), and the GP60 (60 kDa glycoprotein) genes, and two types of DNA staining reagents, ethidium bromide and GelRed<sup>TM</sup>. We determined that one of the tested protocols yields a higher purity of extracted DNA. Additionally, optimised temperatures for the nested PCR of the 18S and GP60 genes were established. Finally, we determined that the GelRed<sup>TM</sup> dye was more sensitive than ethidium bromide, and its low toxicity facilitates handling and disposal and reduces environmental contamination.

scholarly journals LAMP Assay for the Detection of Echinococcus multilocularis Eggs Isolated from Canine Faeces by a Cost-Effective NaOH-Based DNA Extraction Method

Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 847
Author(s):  
Barbara J. Bucher ◽  
Gillian Muchaamba ◽  
Tim Kamber ◽  
Philipp A. Kronenberg ◽  
Kubanychbek K. Abdykerimov ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Dna Extraction ◽  
Sensitivity And Specificity ◽  
Echinococcus Multilocularis ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Cost Effective ◽  
Detection Methods ◽  
Simple Method ◽  
Commercial Kits

The detection of Echinococcus multilocularis in infected canids and the environment is pivotal for a better understanding of the epidemiology of alveolar echinococcosis in endemic areas. Necropsy/sedimentation and counting technique remain the gold standard for the detection of canid infection. PCR-based detection methods have shown high sensitivity and specificity, but they have been hardly used in large scale prevalence studies. Loop-mediated isothermal amplification (LAMP) is a fast and simple method to detect DNA with a high sensitivity and specificity, having the potential for field-application. A specific LAMP assay for the detection of E. multilocularis was developed targeting the mitochondrial nad1 gene. A crucial step for amplification-based detection methods is DNA extraction, usually achieved utilising silica-gel membrane spin columns from commercial kits which are expensive. We propose two cost-effective and straightforward methods for DNA extraction, using NaOH (method 1A) and InstaGeneTM Matrix (method 1B), from isolated eggs circumventing the need for commercial kits. The sensitivity of both assays with fox samples was similar (72.7%) with multiplex-PCR using protocol 1A and LAMP using protocol 1B. Sensitivity increased up to 100% when testing faeces from 12 foxes infected with more than 100 intestinal stages of E. multilocularis. For dogs, sensitivity was similar (95.4%) for LAMP and multiplex-PCR using protocol 1B and for both methods when DNA was extracted using protocol 1A (90.9%). The DNA extraction methods used here are fast, cheap, and do not require a DNA purification step, making them suitable for field studies in low-income countries for the prevalence study of E. multilocularis.

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