Effect of Sample Pooling on the Cycle Threshold Value in RT-PCR Testing for COVID-19 and Logistic Savings

Hiba Sami1, *, Nusrat Perween1, Parvez A. Khan1, Haris M. Khan1
1 Department of Microbiology, Aligarh Muslim University, Aligarh, India

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© 2021 Sami et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at Department of Microbiology, Aligarh Muslim University, Aligarh, India; E-mail:



This ongoing coronavirus disease 2019 (COVID-19) pandemic is a great challenge for our health-care systems and their infrastructure. Diagnostic confirmation of infected individuals based on RT-PCR is important for the containment of viral spread because, despite high viral loads, the infection can be asymptomatic. For overcoming the issue of limited supplies in low socio-economic countries like ours, pooling of specimens was proposed as a method to screen a large number of patients. To analyse the effect of pooling of samples on the sensitivity of RT-PCR, we compared Cycle threshold (Ct) values of pools with those of the deconvoluted (individual) samples.

Materials and Methods:

From March 2020 to October 2020, we performed COVID-19 testing by RT-PCR on samples from areas with varying prevalence of COVID-19 referred to VRDL, JNMCH, Aligarh. For circumventing the limited availability of RNA extraction and PCR reagents, pool testing was started. 5 samples were pooled together, and these pools were tested by RT-PCR. Negative pools were reported as negative, whereas positive pools were deconvoluted, and each sample was tested individually. In this study, 408 pools comprising of 2040 samples were analysed.

Observations and Results:

In our study, a ΔCt value of 0.96 (i.e., an increase in Ct value in the pooled sample as compared to individual sample) was found that shows a slight loss of PCR sensitivity in pooled samples, which is relatively small compared to the inherent clinical sensitivity of the standard assay. But the cost-effectiveness and the ability to reserve resources are considerably high.


As pooling of samples is a cost-effective way for COVID--19 testing, the slight loss in the sensitivity of RT-PCR can be overcome by considering the Ct cut-off value for positive pools slightly above the kit cut off value to circumvent the dilution effect.

Keywords: RT-PCR, Ct value, Cost effectivity, COVID-19, Diagnosis, RNA.