Learn about the differences in molecular testing options for COVID-19. Read more about RT-PCR, RT-LAMP, and RT-NEAR to find the best option for you.

Which NAAT test is best for you, RT-PCR, RT-LAMP, or RT-NEAR?

For SARS-CoV-2 testing, RT-PCR can be highly specific, sensitive, and rapid

Since the beginning of the pandemic, the FDA has issued emergency use authorizations (EUAs) for dozens of COVID-19 tests to meet two needs.

1. To enable screening of thousands of people daily, the FDA authorized high-throughput, lab-based reverse-transcription polymerase chain reaction (RT-PCR) testing. Lab-based RT-PCR can process multiple samples in one run but can take days to provide a result due to shipping and logistical challenges [1].

2. To enable fast individual test results, the FDA authorized point-of-care (POC) tests including rapid RT-PCR tests and isothermal tests such as RT-LAMP (reverse-transcription loop-mediated isothermal amplification) and RT-NEAR (reverse-transcription nicking endonuclease amplification reaction). POC tests are typically designed to be portable and don’t require specialized lab equipment [1].

 

What is the difference between RT-PCR and isothermal tests?

Molecular diagnostic tests, or nucleic acid amplification tests (NAATs), are designed to detect genetic material from a virus. A main difference among approaches is in how the nucleic acid is amplified, which can impact test sensitivity and specificity.

Lab-based RT-qPCR generates billions of copies of a nucleic acid target, making these tests both highly specific and sensitive and particularly suitable for both disease diagnosis and confirming of negative results particularly in symptomatic individuals from another type of test, such as an antigen test [2]. RT-qPCR is broken down into three phases and uses primers and repetitive temperature changes to optimize specificity.

PCR was originally developed in the 1980s and has advanced since then from a manual, time-consuming technique to a more automated process [3]. Most RT-qPCR tests require highly trained operators and well-equipped labs that meet CLIA quality standards, and they can take anywhere from hours to days to provide a result.

RT-LAMP accomplishes amplification using a different method entirely: auto-cycling under isothermal conditions, or under one constant temperature, set by a heat block or water bath. RT-LAMP requires multiple steps with different primers at each step, starting with primers complexing with the DNA target, followed by DNA synthesis and then amplification. While RT-LAMP can provide a result in just a few hours and can be carried out anywhere with basic resources [4], it is a newer technique that is still being assessed in clinical settings [5].

RT-NEAR is another isothermal test that works similarly to RT-LAMP in that it is completed under a constant temperature and with primer sets. Unlike RT-LAMP, in the RT-NEAR process naturally occurring or engineered nicking enzymes, or nicking endonucleases, are used to enable amplification.

What’s the difference between RT-PCR and antigen tests? Read our blog.

Across all testing options, RT-PCR has the highest sensitivity and specificity and is considered the most reliable and accurate [6,7].

Rapid RT-PCR delivers fast, accurate results at the point of care

Rapid RT-PCR delivers sensitive, specific results in CLIA-waived settings. Thermo Fisher Scientific’s Accula SARS-CoV-2 Test uses a proprietary version of PCR, Oscillating Amplification Reaction (OSCAR™), which, in short, lowers the absolute temperature requirements for amplification to reduce cycling times. By innovating on the traditional RT-qPCR process, OSCAR is a derivative of lab-based PCR that can provide results in about 30 minutes versus several hours. The fully automated test uses a palm-sized dock that controls reaction temperatures, timing, and fluid movements within a self-contained test cassette.

Are isothermal amplification and rapid RT-PCR equally sensitive for POC testing?

When assessing sensitivity of POC testing options, one source is most definitive: the FDA’s SARS-CoV-2 Reference Panel compares limits of detection (LoDs) for tests granted EUA. Lower LoD means a test can detect lower levels of virus in a sample, offering a quantitative measure of a test’s analytical sensitivity.

LoD varies by manufacturer, but according to the reference panel LoD range is generally higher for isothermal tests than RT-PCR tests.

The test among lowest LoD on the FDA’s list of tests granted EUA for “rapid” POC testing is a RT-PCR test, the Accula SARS-CoV-2 Test [1].

Rapid, POC RT-PCR and isothermal tests for SARS-CoV-2 [1,8].

Manufacturer

Test

Amplification technology

FDA Reference Panel LoD (NDU/mL)*

Sensitivity limitation noted in IFU**

Thermo Fisher Scientific

Accula SARS-CoV-2

RT-PCR

475 [8]

 

Cepheid

Xpert Xpress SARS-CoV-2

RT-PCR

5,400

 

Roche Molecular Systems

cobas SARS-CoV-2 & Influenza A/B Nucleic Acid Test

RT-PCR

5,400

 

BioFire Diagnostics

BioFire Respiratory Panel 2.1

RT-PCR

6,000

 

Cue Health

Cue COVID-19 Test

Isothermal

60,000

Negative results in asymptomatic individual are presumptive [9]

Abbott Diagnostics

ID NOW COVID-19

RT-NEAR***

(Isothermal)

300,000

Negative results are presumptive [10]

Lucira Health

Lucira CHECK-IT COVID-19 Test Kit

RT-LAMP

(Isothermal)

Not reported

Negative results are presumptive [11]

*NAAT detectable units per mL.
** IFU: Instructions for use.
*** RT-NEAR: Reverse transcription nicking enzyme amplification reaction.

*Cue COVID-19 Test performance was measured with direct swabs. ID Now COVID-19 Test performance was measured with dry swabs. Performance of other tests in the table were measured with swabs in transport media.

What does test sensitivity mean in practical terms when it comes to diagnosing patients?

Sensitivity can impact a test’s ability to detect the virus. False negative results can lead to delay of treatment and could give a false sense of security to individuals leading to increased spread of disease.

Sensitivity also affects whether a test may be used as confirmatory test

Many negative rapid RT-PCR results do not require confirmatory testing. In addition, rapid RT-PCR may be used to confirm an antigen test result, which is not the case with isothermal tests. The CDC advises that “POC NAATs that generate presumptive results are not appropriate for use in confirmatory testing.” [3]

Among other advantages, many rapid RT-PCR tests do not require confirmatory testing

The Infectious Diseases Society of America (IDSA) states isothermal assays are an acceptable testing option when rapid RT-PCR or standard lab-based NAATs are not available, but for most isothermal tests negative results in persons with a high suspicion of SARS-CoV-2 infection should be confirmed with standard NAAT or rapid RT-PCR [12].

While options exist for POC NAATs, one test stands out as a rapid, easy to use solution that offers low LoD – among the lowest on the FDA’s SARS-CoV-2 Reference Panel – and it’s also considered confirmatory: the rapid RT-PCR Accula SARS-CoV-2 Test.

Comparison of rapid SARS-CoV-2 tests

Download our infographic here ›

 

Rapid RT-PCR

Rapid isothermal

(RT-LAMP, RT-NEAR)

Detection target and technology

Viral RNA

Thermal cycling amplification

Viral RNA

Isothermal amplification

IDSA guidelines on confirmatory testing [13]

Not recommended

Recommended

Sensitivity

LOD= 45-54,000 NDU/mL [1,8]

LOD=60,000-300,000 NDU/mL [1]

Detection ability

Early infection

(A low LOD can enable detection of virus before symptoms develop) [13,14,15]

Symptomatic infection

(Higher LOD makes test suboptimal for detection of early infection) [15,16]

Sample method

Nasal swab*

Where testing occurs

Point-of-care

Time to result

25-45 min

5 to 65 min

Result type

Qualitative

*Refer to product specific IFU for details on specimen collection swab

**Not all isothermal tests require confirmatory testing.Data is representative of the majority of authorized tests.

To learn more, visit our Accula SARS-CoV-2 Test page or contact us to request a quote.


Other articles

References:

1. FDA. (2020). SARS-CoV-2 reference panel comparative data.
2. CDC. (2022). Guidance for Antigen Testing for SARS-CoV-2 for Healthcare Providers Testing Individuals in the Community.
3. Smithsonian Institute Archives. The history of PCR.
4. Nature. (2021). A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples.
5. The Centre for Evidence Based Medicine. (2020). What tests could potentially be used for the screening, diagnosis and monitoring of COVID-19 and what are their advantages and  disadvantages?
6. Biocompare. (2022). Choosing an amplification method: PCR vs isothermal.
7. Cleveland Clinic. (2021). COVID-19 and PCR testing.
8. Thermo Fisher Scientific. (2022). Accula SARS-CoV-2 Instructions for Use.
9. Cue COVID-19 Test IFU. (2021). Rev 5.0.
10. Abbott ID NOW COVID-19 IFU. (2021). IN190000 Rev. 8.
11. Lucira CHECK IT COVID-19 Test Kit.  Package Insert (PI) INST019 Rev. C.
12. Clinical Infectious Diseases. (2021). The Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19: molecular diagnostic testing.
13. New England Journal of Medicine. (2020). Presymptomatic SARS-CoV-2 infections and transmission in a skilled nursing facility.
14. Heliyon. (2021). Presymptomatic viral shedding and infective ability of SARS-CoV-2; a case report.
15. bioRXiv (preprint). (2020). SARS-CoV-2 testing: the limit of detection matters.
16. Infectious Disease Society of America. (2020). IDSA guidelines on the diagnosis of COVID-19: molecular diagnostic testing.

 

 

 

 

Disclaimer: 

PRECAUTIONS: This test has not been FDA cleared or approved but has been authorized for emergency use by FDA for use by authorized laboratories; use by laboratories certified under the Clinical Laboratory Improvement Amendments (CLIA) of 1988, 42 U.S.C. §263a, that meet requirements to perform high, moderate or waived complexity tests. The test is authorized for use at the Point of Care (POC), i.e., in patient care settings operating under a CLIA Certificate of Waiver, Certificate of Compliance, or Certificate of Accreditation. The emergency use of this test is only authorized for the duration of the declaration that circumstances exist justifying the authorization of emergency use of in vitro diagnostics for detection and/or diagnosis of COVID-19 under Section 564(b)(1) of the Federal Food, Drug, and Cosmetic Act, 21 U.S.C. § 360bbb-3(b)(1), unless the declaration is terminated or authorization is revoked sooner.

LIMITATIONS: The performance of this test was established based on the evaluation of a limited number of clinical specimens. Clinical performance has not been established with all circulating variants but is anticipated to be reflective of the prevalent variants in circulation at the time and location of the clinical evaluation. Performance at the time of testing may vary depending on the variants circulating, including newly emerging strains of SARS-CoV-2 and their prevalence, which change over time.

Regulatory and Intended Use:
For Use under the Emergency Use Authorization (EUA) Only. For prescription use only. For in vitro diagnostic use.

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