Ningbo First Hospital: Why Continuous Testing Matters

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Section 1: Introduction

COVID-19 or “Coronavirus”, the disease caused by the virus SARS-CoV-2, is the seventh known coronavirus to infect humans since the 2003 SARS outbreak (virus: SARS-CoV). The name SARS is an abbreviation for “severe acute respiratory syndrome”, a type of viral respiratory infection.

In December of 2019, a cluster of pneumonia cases were reported from Wuhan, Hubei Province, China. The disease was traced back to the Huanan Seafood Wholesale Market in Wuhan and subsequently spread like wildfire and sent the globe into a pandemic. Scientists in China were quick to act and sequenced the Novel Coronavirus’ genome in early January. A genome sequence is a complete map of an organism’s DNA or RNA. Having that full sequence allows scientists to identify vital information stored within DNA or RNA, including all of the genes that it might express.

It is important to note here that SARS-CoV-2 is an RNA virus (it doesn’t have DNA) and so any genetic testing that is done in relation to SARS-CoV-2 uses RNA.

Scientists found five genes in the SARS-CoV-2 virus that were deemed as test targets. In order to evaluate the RNA samples, they could either sequence the genome from all patient samples (which would take a very long time), or they could use a method called reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). RT-qPCR is much faster than genome sequencing; it finds the target sections on the RNA from the collected samples in real time and amplifies them into detectable amounts.

Section 2: From DNA to Protein

If you’re interested to learn more about how DNA goes from genetic information to a functional protein keep reading below, otherwise feel free to skip to Section 3: Isolating DNA and Testing with RT-PCR or jump to Section 4: The Case Report.

Within most of our cells lies a nucleus that encapsulates vital genetic information in chromosomes, which are tightly wound structures of DNA. Deoxyribonucleic acid or DNA is a double helix structure made of two strands of nucleic acids. The sequence of these nucleic acids code for certain things like genes.

In the case of animal cells, a gene is a segment of DNA that encodes for a functional piece of RNA (a single strand of ribonucleic acid). If this functional piece of RNA is destined to create a protein, then it will act as an instruction manual for the assembly of a sequence of amino acids that make up a protein. This protein then goes on to carry out its function (see figure 1).

Figure 1. This image shows a simplified version of how DNA turns into RNA which then turns into a protein.3

Unlike human cells, viruses can use either DNA or RNA.  When a DNA-using virus infects a cell, it forces the cell’s own gene copying machines to make more of the virus’ DNA. This DNA is then used to make RNA and, eventually, more viruses. RNA-using viruses bring their own replication machines to the party, and don’t use the host cell’s smarter, more careful machines. Once the genome of an RNA virus is duplicated, it’s immediately ready to be used for protein encoding. Check out this sidebar to see how coronaviruses can infect humans.

For a more in depth look at DNA vs. RNA check out this link. To learn more about the central dogma of DNA and how it encodes for a protein as well as how mutations affect DNA, have a look at this sidebar.

Section 3: Isolating DNA and Testing with RT-PCR

To learn about the methods of how the researchers isolated nucleic acids from the patient’s samples and identified them using RT-PCR keep reading below, otherwise feel free to skip to Section 4: The Case Report.

Nucleic acids were extracted from the patient’s sputum samples (like phlegm), oropharyngeal samples (at the back of the throat), and stool samples. The nucleic acids were isolated in a lab by using a magnetic bead extraction method (see Figure 2 below).

Figure 2. DNA isolation using magnetic beads.4

From here, the nucleic acid sample undergoes reverse transcriptase quantitative polymerase chain reaction (RT-PCR), which exponentially multiplies the DNA to eventually be detected with fluorescence in real time (check out this article that talks about the fundamentals of the RT-qPCR). In order to be successful, the RT-qPCR has to undergo a certain amount of cycles, and for this specific coronavirus, a diagnostic kit created in Shanghai at GeneoDx Biotech was used.

If present, the target DNA will be detected at a certain cycle threshold. A cycle threshold is the cycle number at which the target DNA is detected by fluorescence5. Each cycle is an amplification of DNA. The lower the cycle number the more target DNA is present in the sample. In this diagnostic kit, any cycle threshold above 40 is declared negative, any cycle threshold between 37 and 40 is declared grey zone and must be retested and any cycle threshold result below 37 is a positive test.

This nucleic acid determination of SARS-CoV-2 is aimed at two target genes, the orf1ab gene and the nucleocapsid genes. A positive result for infection by SARS-CoV-2 is determined by the presence of two target genes in the same sample. A single positive is designated as pending, and two negative results is interpreted as the patient being SARS-CoV-2  negative.

The authors suggest that this kit may be more sensitive to amplifying the nucleocapsid gene than the orf1ab gene. That being said, it is worth noting that the method description and collected data within the study is not enough to draw this conclusion. In fact, some of the data in the paper itself (shown below) shows both genes being detected at the same cycle threshold on the same day which casts some suspicion on this suggestion. However if this suggestion is accurate, samples with low viral load might have resulted in a single positive nucleocapsid test. If the medical professionals had complied with the initial criterion that suggests dismissing a single positive gene diagnosis, then the patient’s COVID-19 declaration could have been delayed or missed altogether.

Figure 3 shows the two genes detected through RT-qPCR on day 16, which the cycle threshold values both under 37.

Figure 3. Left, nucleocapsid gene showing signals around cycle 34. Right, orf1ab gene showing signals around cycle 34. Both genes were detected in an effective range under the 37th cycle, which yields a positive result.1

Section 4: The Case Report

This paper discusses a case report for a COVID-19 positive patient in China. It urges medical workers to take symptoms seriously, to not exclude early negative tests and keep testing if there are any suspicions that the patient may have the coronavirus.

The patient was a male and had a six-day history of a fever and cough before he was admitted to the hospital. He had no prior travel history to Wuhan and neither did any of the people he interacted with. His history showed that on January 14, 2020, he had dinner with friends in Ningbo (a city in China). After dinner he went to the hospital for drunkenness, nausea and vomiting.

Three days later, he detected a fever of 38.5˚C (101.3˚F) and on January 18, he went back to the hospital and presented himself to the emergency room with a cough, fever, some chills, a headache, dizziness and muscle ache. He received a blood test and was diagnosed with lymphocytopenia, which is a low level of lymphocytes in the blood, meaning that his immune response was limited. He was given drugs to treat this condition and sent home.

On January 22, his fever persisted so he returned to the hospital. Tests for influenza A and B were both negative but a CT scan (computed tomography scans: a series of X-ray images that provides a cross sectional look at a patient) showed signs of pneumonia in the lower part of his lungs.

Given the recurrent fevers they performed more lab tests with no sign of abnormalities. He was given medication to curb his fever, which worked for roughly three days until his fever came back at 38.9˚C, the highest yet.

Even though there was no contact with ground zero of the COVID-19 outbreak in Wuhan, SARS-CoV-2 RNA was tested for using the RT-qPCR method. The RNA samples were taken from oropharyngeal swabs, which is at the back of the throat where the nasal cavity and the mouth cavity intersect. They also took sputum samples (basically phlegm from the lungs) as well as stool samples.

The tests all came back negative on day 3 and continued to be negative through day 8, even with symptoms. In order for a test to be positive, or at least “pending”, one of the five target genes must be detected in a RT-qPCR test. It wasn’t until day 9 that they detected the “nucleocapsid” gene (a gene that encodes for the protein shell that houses the virus’ genetic information). Since only one of the two target sequences was detected, this patient was designated as ‘pending.’.

With the CT scans on day 7 and the positive nucleocapsid test results on day 9, the patient was designated a suspected COVID-19 case and given antiviral treatment in isolation. Routine blood tests revealed that he also developed a bacterial infection, which they treated with antibiotics. On day 10, he had no signs of fever and on day 12 the patient’s bacterial condition showed signs of improvement. They stopped antibiotics but CT images still showed signs of pneumonia in his upper lungs.

On day 16, the patient’s RT-qPCR tests came back with two different target genes showing positive results: the nucleocapsid gene and the orf1ab gene common in SARS viruses6.

Chest images improved on day 18, with negative RT-qPCR tests on day 19 and 20 and resolved symptoms. He was discharged on day 23 and sent to another hospital for 14 days for monitoring and reported no symptoms. Three more RT-qPCR tests all came back negative.

This all may seem like a positive result and, in reality, it is but we should note the vigilance of these medical professionals. They could have ended all further tests with the consistent negative results that they obtained, but they didn’t. Given all the similarities that this patient had with previous COVID-19-positive patients, they treated him as such and kept testing.

The authors suggest that the patient may have been infected at dinner or at the hospital, based on a typical 3 day incubation period. . His main complaints were fever and cough, the two most common symptoms described in the 1099 COVID-19-positive patients before him. The lymphocytopenia and chest CT abnormalities that he expressed were prevalent in the vast majority of COVID-19 positive patients before him as well. Secondary bacterial infection is thought to have accelerated the replication of the virus and helped move the virus from the deep parts of the lungs to the throat. This case highlights the importance of successive sampling and testing and emphasizes that negative tests may require secondary verification depending on the assay used.. Clinical conditions, routine blood tests and chest images should be highly considered in addition to RNA evidence. This paper also shows how important it is to carefully test any new assay for accuracy before using it for clinical screening. Finally, this is also a strong argument for the continuous development of better and more accurate assays. Tests for viral infection are essential in the fight against SARS-CoV-2. Improvements to those tests will result in a fortified response to the pandemic.


  1. Lv, Ding-Feng, et al. “Dynamic Change Process of Target Genes by RT-PCR Testing of SARS-Cov-2 during the Course of a Coronavirus Disease 2019 Patient.” Clinica Chimica Acta, vol. 506, 2020, pp. 172–175., doi:10.1016/j.cca.2020.03.032.
  2. Genome Research Limited” by Preeti Deshpande is licenced under CC BY-NC-SA.
  3. “Central Dogma of Molecular Biology.” Khan Academy, Khan Academy,
  4. Gane, Andrew. “Magbeads 101: A Guide to Choosing and Using Magnetic Beads.” GE Healthcare Life Sciences, 29 May 2019,
  5. Qiagen. “What Is the Threshold Cycle or Ct Value?” QIAGEN,
  6. “ORF1ab.” UniProt ConsortiumEuropean Bioinformatics InstituteProtein Information ResourceSIB Swiss Institute of Bioinformatics, 11 Dec. 2019,

By Sameer Jafar

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