Combining the capabilities of PCR and NGS to identify and characterize pathogens

Outbreak control and disease prevention efforts rely on swift identification and characterization of novel and existing pathogens, which are then used to develop new and better detection methods. Fast results are crucial for developing effective tools and deploying those tools to limit the spread of an infectious disease. In addition to requiring rapid results, researchers working with challenging sample types (e.g., clinical research and environmental samples that often contain very low copy numbers) need highly sensitive and accurate tools to detect and sequence their target pathogen.

Real-time quantitative PCR (RT-qPCR)- and PCR-based methods are widely used together with next-generation sequencing (NGS) analysis to identify and characterize viral and bacterial pathogens due to their efficiency, sensitivity, and accuracy. RT-qPCR has been used in assays that detect and confirm specific viral sequences obtained using NGS technology. Multiplex PCR has also been used to detect pathogenic bacteria in combination with NGS analysis. These technologies have played key roles in studies involving a variety of viral, bacterial, and mammalian pathogens. Examples include the initial identification and sequencing of the SARS-CoV-2 virus, the identification of Leptospira in a variety of environmental samples in order to understand its mode of transmission, and the identification of mutations in the Epstein-Barr virus (EBV) genome that may cause chronic active EBV (CAEBV) and lymphoma.

Identifying and classifying SARS-CoV-2

The novel coronavirus, SARS-CoV-2 was first identified and classified using a combination of RNA-seq, one-step RT-qPCR, and Rapid Amplification of cDNA Ends (RACE). The authors (Zhu et al. 2020) of the first publication that identified this new RNA virus developed an RT-qPCR screening test that used Takara Bio's One Step PrimeScript RT-PCR Kit (Perfect Real Time) to detect specific viral sequences that were obtained by using RNA-seq to analyze the virus. The authors performed a full-length phylogenetic analysis, which showed that SARS-CoV-2 was similar to some betacoronaviruses detected in bats, but distinct from SARS-CoV and MERS-CoV, two previously identified coronaviruses that can cause severe respiratory illness in humans. 

Another research study (Wu et al. 2020) also used RNA-seq to determine the viral genome sequence of SARS-CoV-2, which they confirmed using RT-qPCR. Next-generation meta-transcriptomic sequencing analysis enabled the researchers to obtain a complete viral genome sequence. Subsequent phylogenetic analysis performed by this group also indicated that the virus is most closely related to a group of SARS-like coronaviruses that had previously been found in bats in China. These analyses were made possible in part by kits and reagents produced by Takara Bio: an RNA library was constructed using our pico-input strand-specific total RNA-seq technology, the viral genome sequence was determined and confirmed by performing RT-qPCR with the One Step PrimeScript RT-PCR Kit (Perfect Real Time), and the SMARTer RACE 5′/3′ Kit was used to study the genome termini.

NGS- and PCR-based methods were utilized in another publication (Zhou et al. 2020) to perform a metagenomic analysis that identified the virus as 96% identical to a bat coronavirus at the whole-genome level. This group also used the SMARTer RACE 5'/3' Kit to determine the 5' ends of the genomes. The pairwise protein sequence analysis of seven conserved nonstructural proteins showed that this virus belongs to the species of SARSr-CoV. They also confirmed that SARS-CoV-2 uses the same cell entry receptor, ACE2, as SARS-CoV, which has proven to be of vital importance in the quest to develop treatments and vaccines to combat the COVID-19 pandemic.

Investigating transmission of leptospirosis in the environment

Leptospirosis is a disease caused by the Leptospira bacteria that infects humans through exposure to the urine of animals carrying these bacteria or contaminated environmental samples. A research group in Okinawa, Japan (Sato et al. 2019) sought to develop new tools to systematically detect Leptospira in order to prevent human infection. They studied the bacterial ecosystem that allows the development of Leptospira during biofilm formation and investigated which animals are potential reservoirs for transmitting this pathogen to humans by analyzing a variety of environmental samples using multiplex PCR and NGS.

The researchers screened environmental water samples from a known endemic region in Japan for rRNA targets specific to Leptospira and animals living nearby. They performed multiplex PCR analysis with Takara Ex Taq HS DNA polymerase to detect bacteria using 16S rRNA targets, which they analyzed by NGS. A similar procedure was carried out using PrimeSTAR HS DNA Polymerase to detect 12S rRNA from vertebrates in the same environmental samples, in order to understand which species are more likely to harbor Leptospira. The presence of certain animals seemed to correlate with high levels of Leptospira, showing a potential link between pathogen and carrier. They were able to draw a correlation between strains of bacteria that propagate pathogenic Leptospira and the animals that are the primary reservoirs of these bacteria. This study demonstrated the robustness of Takara Ex Taq HS and PrimeSTAR HS DNA polymerases to detect bacteria in environmental water samples, which are difficult to work with due to the presence of PCR-inhibitory contaminants. The multiplex PCR method used in the study was a powerful tool that helped determine how Leptospira outbreaks could occur, revealing the interactions between the pathogen, its hosts/carriers, and the environment.

Locating EBV genomic deletions linked to CAEBV-associated lymphomas

Epstein-Barr virus (EBV) is estimated to infect >95% of the population worldwide through the spread of saliva. In most instances, EBV infection will not display any symptoms, or it will lead to infectious mononucleosis before going permanently dormant. However, in some rare cases, the virus remains active, causing CAEBV (chronic active EBV). EBV is an oncogenic herpesvirus that preferentially infects B cells, and less frequently, T and NK cells, causing Hodgkin's lymphoma, Burkitt's lymphoma, epithelial carcinomas, and other malignancies when infecting patients chronically.

Researchers from several Japanese universities carried out a collaborative investigation of the origins of CAEBV and the genomic mutations that lead to the development of tumors and lymphomas (Okuno et al. 2019). They performed long-range PCR with high-fidelity PrimeSTAR GXL polymerase in combination with Sanger and deep sequencing to probe for intragenic deletions in EBV genomes from patients with different CAEBV-associated lymphomas. The researchers identified deletions in microRNA clusters and viral particle production genes which could be linked to the malignancies, but no EBV genomic deletions were found in patients exhibiting infectious mononucleosis. PrimeSTAR GXL polymerase played a vital role in helping to verify the intragenic deletions observed with NGS in this study because it is optimized to perform well with long amplicons and challenging (GC-rich or AT-rich) templates. PCR analysis using a robust, reliable polymerase such as PrimeSTAR GXL is critical for confirming pathogenic mutations, insertions, and deletions observed with NGS.

References

• Okuno, Y. et al. Defective Epstein-Barr virus in chronic active infection and haematological malignancy.  Microbiol. 4, 404–413 (2019). Available at: https://www.nature.com/articles/s41564-018-0334-0
• Sato, Y. et al. Environmental DNA metabarcoding to detect pathogenic Leptospira and associated organisms in leptospirosis-endemic areas of Japan.  Rep. 9, 1–11 (2019). Available at: https://www.nature.com/articles/s41598-019-42978-1
• Wu, F. et al. A new coronavirus associated with human respiratory disease in China. Nature 579, 265–269 (2020). Available at: https://www.nature.com/articles/s41586-020-2008-3
• Zhou, P. et al. Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. bioRxiv01.22.914952 (2020). Available at: https://www.biorxiv.org/content/10.1101/2020.01.22.914952v2

• Zhu, N. et al. A novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. NEJMoa2001017 (2020). doi: https://www.nejm.org/doi/full/10.1056/NEJMoa2001017
  • Zhu et al. supplementary appendix: RT-qPCR protocol and primers used