Sample Collection
This study was approved by the Hospital Ethics Committee, and all study participants were informed. The study population consisted of 733 fever patients at the Second Xiangya Hospital of Central South University, Hunan province, China, between January to February 2020, aged 20 to 70 years. All the patients were diagnosed by two experienced doctors. The samples from the participants were obtained before clinical intensive treatment. Participants who received preoperative radiotherapy or chemotherapy were excluded. Respiratory and stool samples were collected with the consent of all patients. Unless otherwise stated, patients were sampled without gender or age bias. Swabs and stool were stored at -20℃ until use.
RNA extraction and qRT-PCR
Whenever a commercial kit is used, the manufacturer's instructions should be followed without modification. Nucleic acids were extracted by SSNP-2000A Automatic Nucleic Acid Extraction Instrument and nucleic acid extraction kits (Sansure biotech, China). 20 μL extracted nucleic acid sample was added into a PCR amplification tube which contains 30 μL of the prepared PCR-mixed solution, and perform fluorescence quantitative PCR detection on a fluorescent PCR instrument ( detected through fluorescence quantitative PCR using a fluorescent PCR instrument.) Amplification was performed as follows: 50℃ for 30 minutes, 95℃ for 60 seconds, and then 40 cycles, including 95℃ for 15 seconds, 60℃ for 30 seconds.
The primer and probe sequences20:
ORF1b gene
5’-TGGGGYTTTACRGGTAACCT-3’ (Forward)
5’-AACRCGCTTAACAAAGCACTC-3’ (Reverse)
N gene
5′-TAATCAGACAAGGAACTGATTA-3′ (Forward)
5′-CGAAGGTGTGACTTCCATG-3′ (Reverse)
Nucleic acids were analysised Influenza A/B virus nucleic acids test kits (bioPerfectus, China). 5μL extracted nucleic acid sample was added into a PCR amplification tube which contains 20μL of the prepared PCR-mixed solution and performs fluorescence quantitative PCR detection on a fluorescent PCR instrument. Amplification was performed as follows: 50℃ for 30 minutes, 95℃ for 5 minutes, and then 40 cycles, including 95℃ for 10 seconds, 55℃ for 40 seconds. The reference value of the Influenza A virus is 34.7(CT). The reference value of the Influenza B virus is 34.8(CT).
Colloidal gold immunochromatography
The nurse uses a throat swab to apply on both sides of the back wall of the throat or in the nasal cavity. Take a sufficient amount of specimens and put them in a special disposable specimen collection tube for inspection. The specimens should be sent for inspection immediately after collection or stored at 4°C for no more than 3 days. Otherwise, it should be stored below -20°C, and repeated freezing and thawing should not exceed 3 times. According to the Influenza A/B virus antigen detection instructions (Wondfo biotech, China), drop 8 drops of the dilution liquid in the dropper bottle into the plastic tube of the kit, add a sufficient amount of the sample, stir and repeatedly squeeze the tube wall, so that the sample is fully dissolved to the dilution In the liquid. Use a pipette to slowly add 3-4 drops (about 100ul) of the sample solution into the sample hole of the test card, and start recording the time.
Ten respiratory pathogen nucleic acid detection (multiple melting curve method)
The throat swab samples were sucked into the supernatant according to the instructions of the "Nucleic Acid Extraction or Purification Reagents", and the nucleic acid was extracted according to the instructions of the EX-DNA/RNA Virus Nucleic Acid Extraction Kit (Tianlong, China). Configure the RT-PCR system, add 4.4μLRes reaction solution, and 0.6μLRes enzyme solution to a 1.5mL centrifuge tube in turn. Add 25 μL of mineral oil after centrifugation. Place the positive control, negative control and samples on the PCR machine for amplification. Amplification was performed as follows: 50℃ for 30 minutes, 95℃ for 15 minutes, and then 40 cycles, including 94℃ for 30 seconds, 55℃ for 30 seconds, 72℃ for 30 seconds. After the program is run, the instrument will automatically pay to check the Tm value.
Data collection
We reviewed the care records and laboratory test results of fever patients. Admission data for these patients were from January 26th to February 1st, 2020. The two researchers also independently reviewed data collection forms to confirm repeatedly.
Bioinformatics analysis process
The use of different methodological kits to detect SARS-CoV-2 and other common respiratory viruses on samples of fever patients limits our further understanding of SARS-CoV-2 co-infection. We commissioned Sansure to perform RNA metagenomic sequencing on 26 fever patients' samples (SARS-CoV-2 positive: n=16, SARS-CoV-2 negative: n=10 throat swab: n=22, stool: n=4).
The overall process is shown below(Fig.1):
1. Perform a new coronavirus fast test (Fastv) on all data to determine whether it is negative or positive. Compare the virus database to find other co-infected viruses. Compare the bacterial database and annotate the species of the bacteria.
2. Compare to the original data (SortmeRNA) ribosomal database. Split the data into other and rRNA parts.
3. Compare the other part of the reads to hg19. Perform differential analysis of human expressed genes.
4. Compare the other part of the reads with SARS-CoV-2 (NC_045512.2). Analyze virus coverage, depth, mutation, and L/S typing.
5. After excluding the human and SARS-CoV-2 sequences from the other part:
1) Assemble of the 8 throat swabs and 6 stool data (Metaspades). And Remove redundant sequences (Cd-hit). Assess the effect of assembly (Quast).
2) Take the above assembly result as the reference sequence, and do quantitative statistics for all samples (Salmon).
3) Analysis of differences between the negative and positive groups.
4) Perform species and function analysis (Humann3).
Statistical analysis
Continuous variables are expressed as median (IQR) and compared using the Mann-Whitney U test. Categorical variables are expressed as numbers (%) and compared by the chi-square test or Fisher's exact test. A two-sided α of less than 0.05 was considered statistically significant.