Sequential development of several RT‐qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS‐CoV‐2 from influenza A and B

Abstract

Summary Sensitive and accurate RT‐qPCR tests are the primary diagnostic tools to identify SARS‐CoV‐2‐infected patients. While many SARS‐CoV‐2 RT‐qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands‐on‐time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS‐CoV‐2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS‐CoV‐2 RT‐qPCR tests that are originally based on the protocol targeting regions of the RNA‐dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS‐CoV‐2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature‐stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction‐free version enables high sensitivity detection of SARS‐CoV‐2 in about an hour using minimally invasive, self‐collected gargle samples. These RT‐qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS‐CoV‐2 and the most common seasonal influenzas during the vaccination phase of the pandemic.

Vivid COVID-19 LAMP is an ultrasensitive, quadruplexed test using LNA-modified primers and a zinc ion and 5-Br-PAPS colorimetric detection system

“…Unless specified otherwise, LNAs were incorporated using general guidelines established for PCR primers 62 , e.g., targeting 5’ end of primers and avoiding LNA Gs and especially Cs due to their unnaturally strong base pairing conducive to mishybridization and dimer formation. These general principles have been previously shown by our group to improve binding characteristics and specificity for detecting SARS-CoV-2 82 , 122 , 123 .…”

“…9a–h ). Results from our LAMP assays were compared against an RT-qPCR test used for routine screening 82 (distribution of determined Ct values can be seen in Fig. 9c, f, h ).…”

“…We downloaded 500 sequences for SARS-CoV-2 variants (Alpha, Beta, Gamma and Delta) and 4500 for SARS-CoV-2 Omicron variant from the GISAID repository (accessed on March 29 th 2022) and aligned them to the Wuhan reference sequence (NCBI ID: NC_045512.2) using the MAFFT alignment tool (with the parameter - auto) 124 . Next, we filtered out sequences containing ambiguous bases in target primer regions (details can be found on Github ( https://github.com/MultiplexDX/corona_cheks ) 82 ) and called the 90% consensus sequences using SeaView 125 . For in silico cross-reactivity testing, we blasted our LAMP primers against NCBI annotated sequences (GenBank®) selected from a list of high priority pathogens from the same genetic family of SARS-CoV-2 and organisms likely to be present in respiratory/saliva samples (Supplementary Data 3 ).…”

High throughput AS LNA qPCR method for the detection of a specific mutation in poliovirus vaccine strains

“…Here, this AS LNA qPCR is highly suitable for broadening its applicability and adding additional SNPs and targets to the qPCR, due to the characteristics of the LNA probes (increased affinity, increased mismatch discrimination, and the full control of melting point of the hybridization reaction [29] ). Indeed, from these characteristics, LNA-modified nucleotides have been used for a variety of applications, such as in situ hybridization [30] , whole genome amplification [31] , germline SNP genotyping [32] , [33] , viral differentiation SARS-CoV-2 from influenza A and B [34] , as blocker LNA oligonucleotide for the detection of oncogene mutations with high sensitivity, including KRAS and BRAF [35] , and recently as allele-specific primers for accurate and cost-effective diagnosis and quantification of KRAS and BRAF mutations [36] . Here, we are the first to apply this technology for vaccine development screening purposes or vaccine batch release.…”

Viral and Bacterial Profiles in Endemic Influenza A Virus Infected Swine Herds Using Nanopore Metagenomic Sequencing on Tracheobronchial Swabs

“…Still, general care should be taken with Cq values over 36 (<18 genome copy equivalents per 0.1 mL, in our study), as they are difficult to interpret. If the aim is to monitor, screen, or study swIAV in endemic scenarios, RT-qPCR approaches will deliver the highest sensitivity ( 26 , 29 ). Moreover, the (semi-)quantitative nature of metagenomics has previously been demonstrated for swIAV quantification ( 30 , 31 ).…”