Nanosized Fe3O4 an efficient PCR yield enhancer-Comparative study with Au, Ag nanoparticles.

Two real-time quantitative PCR detection systems that contained DNA Green saturated fluorescent dye for detection of Salmonella Enteritidis and Ly Green saturated fluorescent dye for detection of Salmonella spp. were constructed. Furthermore, the specificity of the amplification system increased with the addition of graphene quantum dots. The R2 values of the standard curves were 0.999 (S. Enteritidis) and 0.998 (Salmonella spp.), respectively. The sensitivities of the two amplification systems were evaluated based on three aspects: genomic DNA (1.36 fg/μL, 1.36 fg/μL); bacterial cell without enrichment (2.49 CFU/mL, 2.49 CFU/mL); plasmid containing Gene ID:1828390769 (11.6 copies/μL) and plasmid containing Gene ID:3335471 (1.08 copies/μL). Detection results of fruits and vegetables artificially contaminated with Salmonella showed that the traditional culture method exhibits a lower detection rate than the molecular detection method, and the culture time of the Selective Luria-Bertani broth (SLB) was short. The detection rate was increased compared with that of the Buffered Peptone Water-Selenite Cystine broth (BPW-SC). The two detection systems exhibit high sensitivity and specificity and are suitable for detecting Salmonella in ready-to-eat fruits and vegetables.

Development of a rapid detection method for real-time fluorescent quantitative PCR of Salmonella spp. and Salmonella Enteritidis in ready-to-eat fruits and vegetables

Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour preincubation at 50 °C before real time PCR. Moreover, the results obtained by QD-based HS PCR were comparable to a commercial Taq antibody DNA polymerase. However, no obvious HS effect of QDs was found in real time PCR using Taq DNA polymerase. The findings of this study demonstrated that a cost-effective high-throughput real time PCR based on QD triggered HS PCR could be established with high consistency, sensitivity and accuracy.

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

Quantum dots (QDs) are of great interest due to their unique chemical and physical properties. Recently, a hot start (HS) polymerase chain reaction (PCR) amplification performance based on QDs with a high-fidelity Pfu DNA polymerase has been reported. However, whether QDs can trigger HS effects with other high-fidelity or conventional DNA polymerases is yet to be understood. In the present study, we studied the QD-triggered HS effects with four high-fidelity and three conventional DNA polymerases, and the HS effect comparisons among them were also made. It was found that QDs could trigger a distinct HS PCR amplification performance with all the four tested high-fidelity DNA polymerases, and specific target DNA could be well amplified even if the PCR mixture was pre-incubated for 2 h at 50°C. On the contrary, the HS effects were not prominent with all the three conventional Taq DNA polymerases. Specifically, the fidelity of Pfu is not sacrificed in the presence of QDs, even after a 1 h pre-incubation at 50°C before PCR. Furthermore, the electrophoresis results preliminarily demonstrated that QDs prefer to adsorb high-fidelity polymerases rather than conventional ones, which might result in the QD-triggered HS effects on PCR performance by using high-fidelity DNA polymerases.

/pdf/a-hot-start-alternative-for-high-fidelity-dna-polymerase-7uwkzg3mna.pdf

A hot start alternative for high-fidelity DNA polymerase amplification mediated by quantum dots

It has not been previously reported that the speed of polymerase chain reaction (PCR) can be accelerated by quantum dots (QDs). In the present work, the addition of cadmium telluride (CdTe) QDs resulted in significant time-saving compared with the conventional PCR. The reaction time could be significantly shortened from 143 to 46 min without compromising the general efficiency in a PCR. CdTe QD-facilitated fast PCR also displayed excellent hot-start effects.

CdTe quantum dots accelerate the speed of Pfu-based polymerase chain reaction

Quantitative real-time PCR (qPCR), as an important quantitative technique for nucleic acids, has been widely used in many fields including clinical diagnosis, molecular biology, and cancer research. However, non-specific amplification products are still a frequent problem in qPCR. In this study, we investigated the effects of QDs on real-time amplification based on either SYBR Green I or EvaGreen. It was found that QDs could raise the amplification sensitivity and thus enhance the efficiency using SYBR Green I detection system. In the case of EvaGreen detection systems, addition of QDs also led to a better correlation coefficient than without QDs. EvaGreen-based system gave sharper peaks for melting curves than SYBR Green I. The experiments indicated that the polymerase activity could be partially blocked by QDs at the pre-PCR temperatures, resulting in the improvement of PCR specificity. These results indicated that CdTe QDs could be used as a descent qPCR enhancer. Good amplification fidelity in QDs-facilitated qPCR was also a plus that has not been reported elsewhere.

CdTe quantum dots enhance feasibility of EvaGreen-based real-time PCR with decent amplification fidelity.

Decent hot-start effects were here reported in Taq DNA polymerase-based polymerase chain reaction (PCR) when water-soluble CdTe quantum dots (QDs) were employed. The hot-start effects were revealed by the higher amplicon yields and distinguished suppression of nonspecific amplification after pre-incubation of PCR mix with quantum dots between 30°C and 56°C. DNA targets were well amplified even after PCR mixture was pre-incubated 3 hr at 30°C or 1 hr at 50°C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the polymerase concentration, suggesting that there was an interaction between QDs and Taq DNA polymerase. Moreover, control experiment indicated that hot-start effect is not primarily due to the reduced polymerase concentration resulted from the above interaction. This study provided another good start to investigate potential implications of quantum dots in key molecular biology techniques.

/pdf/quantum-dots-induce-hot-start-effects-for-taq-based-330vuqi633.pdf

Quantum dots induce hot-start effects for Taq-based polymerase chain reaction

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CdTe quantum dots enhance feasibility of EvaGreen-based real-time PCR with decent amplification fidelity.

Quantum dots induce hot-start effects for Taq-based polymerase chain reaction