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Enhancing the specificity and efficiency of polymerase chain reaction using polyethyleneimine-based derivatives and hybrid nanocomposites.

ABSTRACT: There is a general necessity to improve the specificity and efficiency of the polymerase chain reaction (PCR), and exploring the PCR-enhancing mechanism still remains a great challenge. In this paper we report the use of branched polyethyleneimine (PEI)-based derivatives and hybrid nanocomposites as a novel class of enhancers to improve the specificity and efficiency of a nonspecific PCR system. We show that the surface-charge polarity of PEI and PEI derivatives plays a major role in their effectiveness to enhance the PCR. Positively charged amine-terminated pristine PEI, partially (50%) acetylated PEI (PEI-Ac(50)), and completely acetylated PEI (PEI-Ac) are able to improve PCR efficiency and specificity with an optimum concentration order of PEI < PEI-Ac(50) < PEI-Ac, whereas negatively charged carboxyl-terminated PEI (PEI-SAH; SAH denotes succinamic acid groups) and neutralized PEI modified with both polyethylene glycol (PEG) and acetyl (Ac) groups (PEI-PEG-Ac) are unable to improve PCR specificity and efficiency even at concentrations three orders of magnitude higher than that of PEI. Our data clearly suggests that the PCR-enhancing effect is primarily based on the interaction between the PCR components and the PEI derivatives, where electrostatic interaction plays a major role in concentrating the PCR components locally on the backbones of the branched PEI. In addition, multiwalled carbon nanotubes modified with PEI and PEI-stabilized gold nanoparticles are also able to improve the PCR specificity and efficiency with an optimum PEI concentration less than that of the PEI alone, indicating that the inorganic component of the nanocomposites may help improve the interaction between PEI and the PCR components. The developed PEI-based derivatives or nanocomposites may be used as efficient additives to enhance other PCR systems for different biomedical applications.

SUBMITTER: Tong W

PROVIDER: S-EPMC3289439 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Enhancing the specificity and efficiency of polymerase chain reaction using polyethyleneimine-based derivatives and hybrid nanocomposites.

Tong Weiwei W Cao Xueyan X Wen Shihui S Guo Rui R Shen Mingwu M Wang Jianhua J Shi Xiangyang X

International journal of nanomedicine 20120222

There is a general necessity to improve the specificity and efficiency of the polymerase chain reaction (PCR), and exploring the PCR-enhancing mechanism still remains a great challenge. In this paper we report the use of branched polyethyleneimine (PEI)-based derivatives and hybrid nanocomposites as a novel class of enhancers to improve the specificity and efficiency of a nonspecific PCR system. We show that the surface-charge polarity of PEI and PEI derivatives plays a major role in their effec ...[more]

PMID: 22393296

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Project description:Background: Precise determination of amplification efficiency is critical for reliable conversion of within-sample changes in fluorescence occurring on a logarithmic scale to between-sample differences in DNA content occurring on a linear scale. This endeavor is especially challenging for the telomere length (TL) quantitative-PCR (qPCR) assay, where amplification efficiency can vary between reactions targeting telomeric repeats (T) and those targeting a single-copy gene (S) to calculate TL as the T/S ratio. Methods: We compared seven different approaches toward estimating amplification efficiency, including the standard-curve method utilized by the qPCR instrument software, and alternative approaches which estimate efficiency on a reaction-by-reaction basis using the stand-alone program LinRegPCR. After calculating T/S ratios using efficiency estimates from each approach (N = 363), we tested their relative performance on metrics of assay precision and correlates of external validity including chronological age (age range = 1-72 years), across tissues within-person (leukocyte-buccal), and between parents and offspring. Results: Estimated amplification efficiency for telomere reactions was significantly lower than estimates for single-copy gene reactions. Efficiency estimates for both reaction sets were significantly higher when estimated with the standard-curve method utilized by the qPCR instrument relative to estimates reconstructed during the log-linear phase with LinRegPCR. While estimates of single-copy gene efficiency reconstructed using LinRegPCR measured within 90% of perfect exponential doubling (E = 1.92), estimates generated using the standard-curve method were inflated beyond 100% (E = 2.10-2.12), indicating poor fidelity. Despite differences in raw value, TL measurements calculated with LinRegPCR efficiency estimates exhibited similar relationships with external validity correlates to measurements generated using the qPCR instrument software. Conclusion: Since methods to estimate amplification efficiency can vary across qPCR instruments, we suggest that future analyses empirically consider external methods of efficiency calculations such as LinRegPCR, and that already generated data be re-analyzed to glean possible improvements.

Project description:Background and aimDifferent polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and methodsOne hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.ResultsTaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.ConclusionA cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

Dataset Information

Enhancing the specificity and efficiency of polymerase chain reaction using polyethyleneimine-based derivatives and hybrid nanocomposites.

Publications

Enhancing the specificity and efficiency of polymerase chain reaction using polyethyleneimine-based derivatives and hybrid nanocomposites.

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