A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR

In Volume 46 of the Journal of Biosciences, in the article titled Bio Med Frontiers ‘A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR’ by Ridim D Mote, V Shinde Laxmikant, Surya Bansi Singh, Mahak Tiwari, Hemant Singh, Juhi Srivastava, Vidisha Tripathi,Vasudevan Seshadri, Amitabha Majumdar, and Deepa Subramanyam, published on 27 November 2021 (https://doi.org/10.1007/s12038-021- 00231-w), the second author’s name was incorrectly set as V Shinde Laxmikant. The correct name should read as Shinde Laxmikant V.

A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR.

Real-time PCR is a widely used technique for the quantification of gene expression. However, commercially available kits for real-time PCR are very expensive. The ongoing coronavirus pandemic has severely hampered the economy in a number of developing countries, resulting in a reduction in available research funding.
The fallout of this will result in limiting educational institutes and small enterprises from using cutting edge biological techniques such as real-time PCR.
Here, we report a cost-effective approach for preparing and assembling cDNA synthesis and real-time PCR mastermixes with similar efficiencies as commercially available kits. Our results thus demonstrate an alternative to commercially available kits.

Disruptors, a new class of oligonucleotide reagents, significantly improved PCR performance on templates containing stable intramolecular secondary structures.

Intramolecular secondary structures within templates have been shown to lower PCR performance. Whereas many approaches have been developed to mitigate such impairment on PCR, their effects can vary greatly depending on template sequences.
  1. Here we present a novel, universally effective approach to improve PCR performance involving specifically designed oligonucleotides called disruptors. Our Provider A disruptor contained three functional components, an anchor designed to initiate template binding, an effector to disrupt the intramolecular secondary structure, and a 3′ blocker to prevent its elongation by DNA polymerase.
  2. A functional mechanism for a disruptor to improve PCR efficiency was proposed where anchor first binds to template followed by effector-mediated strand displacement to unwind intramolecular secondary structure. Such a mechanism was consistent with the observation that anchor played a more critical role for disruptor function.
  3. As an example of potential disruptor applications, inverted terminal repeat sequences of recombinant adeno-associated virus vectors were successfully amplified in the presence of disruptors despite their well-known reputation as some of the most difficult templates for PCR amplification and Sanger sequencing due to their ultra-stable T-shaped hairpin structures.
  4. In stark contrast, both DMSO and betaine, two PCR additives routinely used to facilitate PCR amplification and Sanger sequencing of GC-rich templates did not demonstrate any improving effect.

Optimizing the assignment of swabs and reagent for PCR testing during a viral epidemic.

Early large-scale swab testing is a fundamental tool for health authorities to assess the prevalence of a virus and enact appropriate mitigation measures during an epidemic.
The COVID-19 pandemic has shown that the availability of chemical reagent required to carry out the tests is often a bottleneck in increasing a country’s testing capacity.
Further, demand is unevenly spread between more affected regions (which require more tests they can perform) and less affected ones (which have spare capacity).
These issues hint at the opportunity of increasing test capacity via the optimal allocation of swabs and reagent to laboratories.
We prove that this is the case, proposing an Integer Programming formulation to maximise the number of tests a country can perform and validating our approach on both real-life data from Italy and synthetic instances.
Our results show that increased inter-regional collaboration and a steadier supply of reagent (i.e., coming from local production sites rather than international shipments) can dramatically increase testing capacity.
Accordingly, we propose short-term and long-term recommendations for policy makers and health authorities.

Development of a novel quantitative real-time PCR assay with lyophilized powder reagent to detect African swine fever virus in blood samples of domestic pigs in China.

  • African swine fever (ASF) is a devastating disease, which is causing huge economic losses in China. Therefore, it is urgent to provide a rapid, highly specific and sensitive diagnostic method for the detection of African swine fever virus (ASFV), the ASF infectious agent.
  • In this study, a novel quantitative real-time polymerase chain reaction (qPCR) assay with lyophilized powder reagents (LPR), targeting the major structural protein p72 gene, was established for the detection of ASFV. This assay had many advantages, such as saving time and money, good sensitivity and repeatability.
  • The sensitivity of this assay was 100 copies/μl of ASFV plasmid templates, and the assay showed 10-fold greater sensitivity than a qPCR assay recommended by OIE.
  • Furthermore, specificity analysis showed that the qPCR with the LPR for ASFV had no cross-reactivity with other important swine pathogens.
  • In clinical diagnoses of 218 blood samples of domestic pigs in China, the positive rate of the diagnosis of ASFV by qPCR with the LPR and commercial kit reached 80.73% (176/218) and 76.61% (167/218), respectively. The coincidence rate between the two assays is 92.20% (201/218), and kappa value is 0.768 (P < 0.0001) by SPSS analysis.
  • The overall agreement between the two assays was 95.87% (209/218). Further Pearson correlation and linear regression analysis showed a significant correlation between the two assays with an R2 value of 0.9438. The entire procedure, from specimen processing to result reporting, can be completed within 2 hours. Our results demonstrated the qPCR-LPR assay is a good laboratory diagnostic tool for sensitive and efficient detection of ASFV. This article is protected by copyright. All rights reserved.

Identification and removal of contaminating microbial DNA from PCR reagents: impact on low-biomass microbiome analyses.

Reagent-derived contamination can compromise the integrity of microbiome data, particularly in low microbial biomass samples. This contamination has recently been attributed to the ‘kitome’ (contamination introduced by the DNA extraction kit), prior to which attention was mostly paid to potential contamination introduced by PCR reagents.
In this study, we assessed the proportion to which our DNA extraction kit and PCR master mix introduce contaminating microbial DNA to bacterial microbial profiles generated by 16S rRNA gene sequencing.
Utilizing a commercial dsDNase treatment protocol to decontaminate the PCR master mix, we demonstrated that the vast majority of contaminating DNA was derived from the PCR master mix.
Importantly, this contamination was almost completely eliminated using the simple dsDNase treatment, resulting in a 99% reduction in contaminating bacterial reads.
We suggest that dsDNase treatment of PCR reagents should be explored as a simple and effective way of reducing contamination in low-biomass microbiome studies and producing more robust and reliable data.
Reagent contamination with microbial DNA is a major problem in microbiome studies of low microbial biomass samples. Levels of such contaminating DNA often outweigh what is present in the sample and heavily confound subsequent data analysis. Previous studies have suggested this contamination is primarily derived from DNA extraction kits.

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Here, we identified the PCR master mix as the primary source of contamination and showed that enzymatic removal of the contamination drastically reduced the blank signal and improved precision. Decontamination of PCR master mixes may have the potential to improve the sensitivity and accuracy of low-biomass microbiome studies.

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