Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

Sang, Fuming; Yang, Yang; Lin, Yuan; Ren, Jicun; Zhang, Zhizhou

doi:10.1039/c5nr03596a

Cited by 15 publications

(10 citation statements)

References 28 publications

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“…To reduce PCR errors caused by primer dimers and/or nonspecific fragments, which may occur in multiplex-PCR amplification, we used Pfu-DNA polymerase with lower error rate than taq polymerase and limited the pre-amplification step of the first PCR to 13 cycles. (32,33) We compared the conventional PCR protocol with a two-stage PCR protocol using the same primers and cycle and amplification conditions. The quality values of the amplicons produced by the two-stage and conventional PCR were compared using the Sanger method.…”

Section: Discussionmentioning

confidence: 99%

A sensitive method for low input amount of exosomes in mouse model liquid biopsy using two-stage PCR

Rho¹,

Choi²,

Park³

et al. 2020

Preprint

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Background Recently, many potential non-invasive biomarkers have been developed using exosome-based liquid biopsy for early detection, diagnosis, and treatment of cancer. However, exosome analyses from small liquid biopsy samples have been limited with regard to sensitivity and efficiency. Methods We used a breast cancer model mouse with ERBB2 overexpression and collected liquid biopsy samples. We performed two-stage PCR for ERBB2, PPP1R1B, GRB7, and STARD3 genes. We compared conventional PCR methods with our two-stage PCR method. Both methods had same step of primer and cycle and amplification condition for RT-qPCR. PCR amplicons quality was validated by using the Sanger method. Results Quantitative analysis of all samples by conventional PCR compared with two-stage PCR template dilution (1/200) showed similar Ct values in all sample groups (cell, supernatant, urine, and plasma). Sequencing of the two-stage PCR-derived products revealed no changes in product sequence compared to that of conventional PCR. The obtained sequences showed 98-99% match of target gene sequences in the databases according to BLAST searches. Conclusion Two-stage PCR has better sensitivity and efficiency than conventional PCR methods for small-volume samples. Additionally, this method is useful for monitoring, as many genes can be assayed at once.

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Section: Discussionmentioning

confidence: 99%

A sensitive method for low input amount of exosomes in mouse model liquid biopsy using two-stage PCR

Rho¹,

Choi²,

Park³

et al. 2020

Preprint

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“…Thus, a highly sensitive, specific, and rapid method of detecting FMDV is required. Nanomaterial-assisted PCR (nano-PCR) has recently been developed to overcome these limitations and improve efficiency over conventional PCR [ 17 , 18 , 19 ]. Nanomaterials, including gold and magnetic nanoparticles, titanium oxide, quantum dots, and carbon-based materials have been used to develop PCR assays with outstanding sensitivity, specificity, and efficiency.…”

Section: Introductionmentioning

confidence: 99%

Effects of Graphene Oxide-Gold Nanoparticles Nanocomposite on Highly Sensitive Foot-and-Mouth Disease Virus Detection

Kim

Lee

et al. 2020

Nanomaterials

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The polymerase chain reaction (PCR) has become a powerful molecular diagnostic technique over the past few decades, but remains somewhat impaired due to low specificity, poor sensitivity, and false positive results. Metal and carbon nanomaterials, quantum dots, and metal oxides, can improve the quality and productivity of PCR assays. Here, we describe the ability of PCR assisted with nanomaterials (nano-PCR) comprising a nanocomposite of graphene oxide (GO) and gold nanoparticles (AuNPs) for sensitive detection of the foot-and-mouth disease virus (FMDV). Graphene oxide and AuNPs have been widely applied as biomedical materials for diagnosis, therapy, and drug delivery due to their unique chemical and physical properties. Foot-and-mouth disease (FMD) is highly contagious and fatal for cloven-hoofed animals including pigs, and it can thus seriously damage the swine industry. Therefore, a highly sensitive, specific, and practical method is needed to detect FMDV. The detection limit of real-time PCR improved by ~1000 fold when assisted by GO-AuNPs. We also designed a system of detecting serotypes in a single assay based on melting temperatures. Our sensitive and specific nano-PCR system can be applied to diagnose early FMDV infection, and thus may prove to be useful for clinical and biomedical applications.

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“…Currently, the commonly used method for bacteria detection is the conventional culture-based method, however, it suffers from 3–5 days for obtaining results . Although enzyme-linked immunosorbent assay (ELISA) , and polymerase chain reaction (PCR) , have been developed as replacements for the culture method, they are limited to tedious procedures and poor efficiency and are not as yet user-friendly for average users who have limited training in chemical or biological laboratories. They are also limited to intensive infrastructures. , In recent years, numerous approaches have been developed for pathogen detection such as lateral flow immunoassay (LFIA), , optical biosensor, , surface enhanced Raman scattering biosensor, , and electrochemical biosensor. , However, among these methods, only LFIA can meet the requirements of simple, rapid, and point-of-care (POC) detection simultaneously.…”

Section: Introductionmentioning

confidence: 99%

“…Currently, the commonly used method for bacteria detection is the conventional culture-based method, however, it suffers from 3−5 days for obtaining results. 6 Although enzyme-linked immunosorbent assay (ELISA) 7,8 and polymerase chain reaction (PCR) 9,10 have been developed as replacements for the culture method, they are limited to tedious procedures and poor efficiency and are not as yet userfriendly for average users who have limited training in chemical or biological laboratories. They are also limited to intensive infrastructures.…”

Section: ■ Introductionmentioning

confidence: 99%

Colorimetric-Fluorescent-Magnetic Nanosphere-Based Multimodal Assay Platform for Salmonella Detection

et al. 2018

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Rapid and sensitive foodborne pathogen detection assay, which can be applied in multiple fields, is essential to timely diagnosis. Herein, we proposed a multisignal readout lateral flow immunoassay for Salmonella typhimurium (S. typhi) detection. The assay employs colorimetric-fluorescent-magnetic nanospheres (CFMNs) as labels, which possess multifunctional target separation and enrichment, multisignal readout, and two formats of quantitation. The assay for S. typhi detection involves magnetic separation and chromatography. First, the S. typhi were separated and enriched from matrix by antibody labeled CFMNs, and then the S. typhi-containing suspension is added onto the sample pad to flow up the test strip. The introduction of magnetic separation enhances anti-interference ability and 10-fold sensitivity, making the assay possible for practical application. The assay has realized naked eye detection of 1.88 × 104 CFU/mL S. typhi, and 3.75 × 103 CFU/mL S. typhi can be detected with a magnetic assay reader, which is 2–4 orders of magnitude lower than other label-based LFIAs, with a quantitation range of 1.88 × 104 to 1.88 × 107 CFU/mL by measuring the fluorescence intensity and magnetic signal. Moreover, the successful detection of S. typhi in complex matrix (tap water, milk, fetal bovine serum, and whole blood) indicated its potential application in real samples.

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Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

Cited by 15 publications

References 28 publications

A sensitive method for low input amount of exosomes in mouse model liquid biopsy using two-stage PCR

A sensitive method for low input amount of exosomes in mouse model liquid biopsy using two-stage PCR

Effects of Graphene Oxide-Gold Nanoparticles Nanocomposite on Highly Sensitive Foot-and-Mouth Disease Virus Detection

Colorimetric-Fluorescent-Magnetic Nanosphere-Based Multimodal Assay Platform for Salmonella Detection

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