Development and Evaluation of Single-tube Nested PCR (STNPCR) for the Detection of Porcine Circovirus type 2 (PCV2)

Pontes, Nayara Elis Cabral; Barbosa, Clara Nilce; Jesus, André Luiz Santos de; Silva, J. G.; Freitas, Amanda de Lira

doi:10.1111/tbed.12022

Cited by 8 publications

(6 citation statements)

References 23 publications

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“…STNPCR employed in this study has been successfully applied for detection of other pathogenic microorganisms such as Schistosoma mansoni [42, 46], Plasmodium falciparum [47], Yersinia pestis [48], dengue virus serotypes [26], Vibrio cholera O1 [27], Leishmania chagasi [25], Porcine Circovirus type 2 [28] and Mycobacterium tuberculosis [29], causing range of clinical conditions.…”

Section: Discussionmentioning

confidence: 99%

“…The sample size required for the study was calculated according to following standard formulas, [TP + FN = Z 2 × (SN(1-SN))/W 2 and n = (TP + FN)/P where TP: true positive, FN: false negative, SN: lowest acceptable sensitivity of diagnostic test used; According to previously published data, STNPCR method was 100% sensitive for diagnosis ([28]. Therefore SN was taken as 98.0% for sample calculation, Z: normal distribution value at particular confidence interval (i.e.…”

Section: Methodsmentioning

confidence: 99%

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A highly sensitive modified nested PCR to enhance case detection in leishmaniasis

et al. 2019

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Background Human leishmaniasis is one of the major parasitic diseases with worldwide distribution. Sri Lanka is a recently established focus of leishmaniasis caused by a variant Leishmania donovani . Early case detection and management is a main approach identified for L. donovani control in the regional leishmaniasis elimination drive. Usefulness of light microscopy and in-vitro culture are limited in chronic, atypical or treated lesions though timely and accurate detection of all light microscopy/in-vitro culture negative cases of all forms of leishmaniasis is necessary for treatment. Timely treatment is important to minimize risk for death in visceral disease and undesired sequelae of long standing infection and illness on both patients and community. We described a 100% sensitive, Leishmania spp. specific modified version of a nested PCR (Mo-STNPCR) that also minimizes carry over and cross contaminations while facilitate investigation of light microscopy and in-vitro culture negative clinically suggestive cases of leishmaniasis. Methods Leishmania DNA was amplified using previously published P221: 5′-GGTTCCTTTCCTGATTTACG-3′ and P332: 5′-GGCCGGTAAAGGCCGAATAG-3’outer primers followed by a nested reaction using P223: 5′-TCCCATCGCAACCTCGGTT-3′ and P333: 5′-AAGCGGGCGCGGTGCTG-3′ inner primers that by passes the requirement of tube handling between the two steps of the conventional nested PCR. Leishmania DNA was detected in a range of infected tissue material. Infected material from patients with cutaneous leishmaniasis ( n = 30), visceral leishmaniasis ( n = 10) and from a control group including patients with non-leishmanial skin diseases ( n = 10), other systemic diseases ( n = 10) and healthy individuals ( n = 10) were examined with Mo-STNPCR. Results were further compared with those of light microscopy and in-vitro culture. Results Mo-STNPCR method was 100% sensitive and 100% specific for diagnosis of leishmaniasis. Light microscopy and in-vitro culture were positive in 75.0% ( n = 30/40) and 72.5% ( n = 29/40) samples respectively where combined results of them gave 87.5% ( n = 35/40) sensitivity. Mo-STNPCR did not cross react with control samples. Furthermore, Mo-STNPCR reduces the risk of cross-contaminations and carry over contaminations since the full reaction is carried out without opening the tubes. Per patient cost was calculated as 22 USD while the same was 3 and 6 USD for light microscopy and in-vitro culture respectively. Conclusion Mo-STNPCR method is a useful tool in detecting leishmaniasis in minority of cases that go undetected by first line investigations. ...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A highly sensitive modified nested PCR to enhance case detection in leishmaniasis

et al. 2019

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“…With the rapid development of molecular biology techniques, researchers have established several diagnostic methods for pathogens detection. As standard and conventional testing techniques, polymerase chain reaction (PCR) and quantitative PCR (qPCR) techniques have been widely employed by laboratories and firms, because of their rapid, specific, and sensitive characteristics (23)(24)(25). However, these approaches require expensive equipment and are inconvenient for field inspection.…”

Section: Introductionmentioning

confidence: 99%

Development of Recombinase Aided Amplification Combined With Disposable Nucleic Acid Test Strip for Rapid Detection of Porcine Circovirus Type 2

Chen

Fan

et al. 2021

Front. Vet. Sci.

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Porcine circovirus type 2 (PCV2) is the dominant causative agent of PCV2 systemic disease (PCV2-SD) in pigs. It can also associate with other diseases such as respiratory and enteric diseases, reproductive failure, porcine dermatitis and nephropathy syndrome in pigs. Currently, PCV2 infection is a considerable threat in the swine industry. Therefore, it is of great significance to prevent, control, and accurately detect PCV2 in pig farms. Recombinase aided amplification (RAA) technology is an isothermal nucleic acid amplification technology that could rapidly amplify the target gene fragment at a constant temperature. The amplification products labeled with specific molecules could be visually detected using the test strip with the corresponding antibody. In the present study, the RAA technology combined with a nucleic acid test strip (RAA-strip) was established for simple and specific detection of PCV2. Primers and probes targeting the PCV2 ORF2 gene were designed according to the RAA technology principles. The PCV2 RAA-strip established in this study could detect as low as 103 copies/μL of recombinant plasmids containing the PCV2 ORF2 gene fragment. The lowest detection limit about viral DNA and virus titers was 6.7 × 10−6 ng/μL and 10 TCID50/mL, respectively. Furthermore, no cross-reaction with other porcine viruses occurred at 37°C and within 15 min. We used 42 clinical samples to assess the performance of our established method. The positive rate of clinical samples detected by PCV2 RAA-strip was 50.00%. This was similar to that detected by PCV2 PCR (45.24%). In conclusion, due to the advantages of strong specificity, high sensitivity, excellent reproducibility, and simple operation method, our PCV2 RAA-strip is suitable for the rapid clinical detection of PCV2 on-site.

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“…Next, an aliquot of the amplified product serves as the template in the running of a second PCR using an inner set of primers [3,4,5,6]. Typically, the first round with low number of cycles (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) serves to boost the scarce amount of the natural DNA template to generate a sufficient amount of a synthesized template for the second round. In contrast to single round PCR, nPCR is a very sensitive assay to detect extremely low copy number target DNA.…”

Section: Introductionmentioning

confidence: 99%

Improving sensitivity of single tube nested PCR to detect fastidious microorganisms

Shatleh-Rantisi

Tamimi

Ashhab

2020

Heliyon

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Single Tube Nested PCR (ST-nPCR) is of value to clinical laboratories with limited settings for the detection of fastidious microorganisms. The detection sensitivity of ST-nPCR is dependent on ensuring minimal leftovers of outer primers during the second round of the reaction. In this work, we investigated various approaches to optimize the performance of outer primers, including decreasing outer primer concentrations; using antisense oligonucleotides to block outer primers; using chemically modified inner primers; and using Q5 Taq polymerase that lacks 5 0 -3 0 exonuclease and strand displacement capabilities. These solutions were tested on C. abortus and C. psittaci, which are both fastidious intracellular bacteria that are difficult to diagnose.The best obtained result was by using Q5 Taq polymerase. A detection limit with a range between 0.1 and 1 ag was achieved, which corresponds to a range between 0.2 and 2 copies of the plasmid positive control. This level of sensitivity is comparable or even better than the sensitivity achieved by TaqMan probe based real-time PCR assays. The assay was validated using 70 veterinary clinical samples from small ruminant abortions and 10% of these samples gave positive results.In conclusion, sensitivity of ST-nPCR to detect fastidious microorganisms can be improved by using Taq polymerases that lacks 5 0 -3 0 exonuclease. The proposed assay is affordable and applicable to a wide range of fastidious pathogens and can be suitable for laboratories with limited settings.

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Development and Evaluation of Single-tube Nested PCR (STNPCR) for the Detection of Porcine Circovirus type 2 (PCV2)

Cited by 8 publications

References 23 publications

A highly sensitive modified nested PCR to enhance case detection in leishmaniasis

A highly sensitive modified nested PCR to enhance case detection in leishmaniasis

Development of Recombinase Aided Amplification Combined With Disposable Nucleic Acid Test Strip for Rapid Detection of Porcine Circovirus Type 2

Improving sensitivity of single tube nested PCR to detect fastidious microorganisms

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