Nucleic Acid Detection

Nucleic Acid Detection


For infectious diseases, rapid and accurate identification of the pathogen is critical for effective management and treatment, but diagnosis remains a challenge, particularly in resource-limited areas. Methods that accurately detect nucleic acids from pathogens can provide robust, accurate, rapid, and ultra-sensitive technologies for the diagnosis of pathogens at the point of care and thus generate information that is invaluable for disease management and treatment. Several technologies, mostly based on PCR, have been used for the detection of pathogens; however, these require expensive reagents and equipment, and qualified personnel.

CRISPR / Cas systems have been used for genome editing, based on their ability to accurately recognize and cleave specific DNA and RNA sequences. In addition, upon recognition of the target sequence, certain CRISPR / Cas systems, including the Cas13, Cas12a and Cas14 orthologs, exhibit collateral nonspecific catalytic activities that can be used for nucleic acid detection, for example, by degradation of a labelled nucleic acid to produce a fluorescent compound. sign. CRISPR / Cas systems can be multiplexed, allowing a single diagnostic test to identify multiple targets down to attomolar concentrations (10–18 mmol / L) of target molecules.

The development of devices that combine CRISPR / Cas with lateral flow systems can enable cost-effective, accurate and highly sensitive field-deployable diagnostics. These sensors have countless applications, from human health to agriculture. In this review, we discuss recent advances in the field of CRISPR-based biosensing technologies and highlight ideas of its potential use in a wide variety of applications.

Importance of SARS-COV-2 Nucleic Acid Testing

Commonly used clinical methods to detect viral pathogens include virus isolation and viral nucleic acid testing (NAT). Isolation of the virus is the “gold standard” for laboratory diagnosis, but it is far from meeting the clinical needs required for a large number of suspected patients in a short period of time. Furthermore, virus isolation can only be performed in laboratories of biosafety level 3 (BSL-3) or higher; Conventional labs do not meet these requirements, making it challenging to confirm a diagnosis. NAT testing procedures for RNA viruses include RNA extraction, nucleic acid amplification, and target gene detection. Polymerase chain reaction (PCR) or isothermal amplification can be used for nucleic acid amplification.

Isothermal can be divided into many types, such as LAMP (loop-mediated isothermal amplification) and RPA (recombinase polymerase amplification). RPA is used in the SHERLOCK (CRISPR-cas13) system, while LAMP is used for amplification in the DETECTOR (CRISPR-cas12) assay. A specific sequence or sequences of the SARS-CoV-2 genome are amplified and detected with fluorescence-labelled probes using the quantitative real-time reverse transcription-PCR (qRT-PCR) technique, during which viral loads of the patients were detected. Detection by qRT-PCR is the most sensitive, specific and simple diagnostic tool available today.

As confirmed in the first six versions of the Novel Coronavirus Pneumonia Diagnosis and Treatment Protocol, published by the National Health Commission of China, a diagnosis of COVID-19 can be confirmed by two approaches: (1) evaluation of results NAT positives by real quantitative fluorescence -PCR reverse transcription in time (qRT-PCR) and (2) genome sequencing evaluating high homology with SARS-CoV-2. Clinically, qRT-PCR is used to confirm the diagnosis in most suspected cases. Compared to gene sequencing, qRT-PCR is faster, can be used on a larger scale, and is more affordable. As the pandemic progresses, a variety of SARS-CoV-2 NAT kits have been rapidly developed in China, most of which are based on qRT-PCR technology.

NAT Existing Issues for SARS-COV-2

Although qRT-PCR tests had many of the advantages listed above, their results are subject to many influencing factors. Since the COVID-19 outbreak, NAT tests, which are considered the “gold standard” of diagnosis, sometimes generate false-negative results due to many factors, which has affected early diagnosis of the disease and posed a major challenge for disease mitigation and containment. pandemic. The main limitations of SARS-CoV-2 NAT are discussed below.

  • NAT kits are technically new

After the emergence of a new virus, a certain period of time is required to develop a validated kit through isolation, sequencing and identification. As awareness of the disease increases, the kits are gradually optimized, resulting in greater specificity and precision. The detection sensitivity of most qRT-PCR kits is between 100 and 500 viral copies in one reaction. As such, kits can only be used to detect high viral loads, and detection results can be significantly inconsistent between different kits manufactured or even between different lots of kits from the same manufacturer.

Due to the need to contain the pandemic, SARS-CoV-2 NAT kits can be used directly to diagnose COVID-19 before they are validated through a large number of clinical trials, and the reproducibility of such kits is problematic. Furthermore, most false-negative patients are in the early stage of the disease. According to existing reports, the incubation period for COVID-19 is up to 14 days and, in exceptional cases, it can reach 38 or 40 days (15, 16). The early clinical manifestations of COVID-19 are not typical. Once the disease enters the advanced stage, it progresses very rapidly. In the early stage of infection, when the viral copy number is below the detection threshold of qRT-PCR, false-negative results are inevitable.

  • Many specimens are not kept in optimal conditions

According to China’s regulations on the management of infectious diseases, samples from positive cases must be sent to the Center for Disease Control and Prevention to confirm the diagnosis; however, long-term transport of samples is likely to cause degradation of viral RNA. As such, China ultimately allowed these samples to be analyzed by the testing department of any medical institution, as long as the testing department is equipped with a qualified clinical PCR laboratory and passes virus containment inspection. However, before the outbreak of this pandemic, most clinical laboratories were operating to meet clinical needs, and only a few had additional testing capacity.

When the number of suspected cases reaches 100,000 and the number of daily tests exceeds the laboratory’s testing capacity, testing should be postponed on some samples. It is difficult to send samples for analysis in a timely manner, and a significant number of samples must be delayed until the maximum allowable delay time is reached before testing. Also, some samples are so delayed that the optimal time window for testing is missed. Today, with the rapid expansion and renovation of laboratories, the sample transportation and analysis process has improved significantly.