CN111549183A - Method for simultaneously detecting SARS-CoV-2 virus and other pathogens in plasma of convalescent coronary pneumonia convalescent patient - Google Patents

Abstract

The invention relates to a method for simultaneously detecting SARS-CoV-2 virus and other pathogens in plasma of a convalescent patient with new coronary pneumonia, wherein a reaction system used by the method comprises the following steps: (1) a primer group for detecting SARS-CoV-2 virus, (2) a detection primer group for detecting various other pathogens, (3) necessary nucleic acid extraction reagent and color reagent; (4) internal quality control of nucleic acid samples, amplification quality control and positive quality control. The simultaneous detection of SARS-CoV-2 virus and respiratory tract pathogen in the convalescent plasma of the new coronary pneumonia convalescent patient is as follows: after one-time nucleic acid extraction, the sample is subjected to two real-time fluorescence PCR detections, and SARS-CoV-2 virus and various pathogenic microorganisms in the sample are respectively determined.

Description

Method for simultaneously detecting SARS-CoV-2 virus and other pathogens in plasma of convalescent coronary pneumonia convalescent patient

Technical Field

The invention belongs to the field of medical biotechnology, and particularly relates to a method for simultaneously detecting SARS-CoV-2 virus and other pathogens in plasma of a convalescent patient with new coronary pneumonia.

Background

The novel Coronavirus pneumonia (Coronavir Disease 2019, COVID-19) is an Acute infectious Disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. SARS-CoV-2 is a enveloped single-stranded positive-strand RNA virus, a coronavirus of the family Coronaviridae, the family Beta, the seventh member of the family of human coronavirus of the present infection, and is transmitted mainly through respiratory droplets and intimate contact, and possibly through the aerosol and digestive tract faecal-oral pathways. The COVID-19 is clinically mainly manifested by fever, dry cough and hypodynamia, some patients can have symptoms such as dyspnea, diarrhea and the like, can progress to symptoms such as acute respiratory distress syndrome, septic shock, blood coagulation dysfunction and the like, and serious patients can cause the death of the patients.

Clinical experiments on therapeutic drugs and antiviral vaccines of COVID-19 are being conducted in various countries. Clinical symptoms are classified according to COVID-19: light, normal, heavy and critical types. In the diagnosis and treatment scheme for pneumonia infected by novel coronavirus (trial sixth edition), recovery plasma treatment of convalescent patients is formally listed as a treatment method for severe and critical cases, and is mainly suitable for patients with severe and severe new coronary pneumonia with fast disease progression.

In the preparation of blood products using plasma of convalescent people as raw material, it is necessary to strictly detect virus and various other infectious agents in the plasma of convalescent people, so a quick and efficient method for in vitro quality detection of the plasma of convalescent people is needed to quickly detect SARS-CoV-2 virus and other pathogenic bacteria in the plasma.

Disclosure of Invention

The invention firstly relates to a detection kit for simultaneously detecting SARS-CoV-2 virus and a plurality of other pathogens in a plasma sample, the kit comprises:

(1) a primer group for detecting SARS-CoV-2 virus,

(2) a detection primer group for detecting a plurality of other pathogens,

(3) necessary nucleic acid extraction reagent and color reagent.

The primer group for detecting SARS-CoV-2 virus is two groups of fluorescent PCR primer groups as follows:

SARS-CoV-2 target one (orf1ab) TEXAS RED610-BHQ2 fluorescence signal group

SEQ ID NO. 1: forward primer (F): CCCTGTGGGTTTTACACTTAA

SEQ ID NO. 2: reverse primer (R): ACGATTGTGCATCAGCTGA

SEQ ID NO. 3: fluorescent probe (P): CCGTCTGCGGTATGTGGAAAGGTTATGG, the 5 'end of the probe is TEXAS RED610, and the 3' end is BHQ 2;

SARS-CoV-2 target two (N) FAM-BHQ1 fluorescence signal set

SEQ ID NO. 4: forward primer (F): GGGGAACTTCTCCTGCTAGAAT

SEQ ID No. 5: reverse primer (R): CAGACATTTTGCTCTCAAGCTG

SEQ ID NO. 6: fluorescent probe (P): TTGCTGCTGCTTGACAGATT, the 5 'end of the probe is FAM, and the 3' end is BHQ 1;

the primer group for detecting a plurality of other pathogens is

1) ROX-BHQ1 fluorescence signal set

Respiratory syncytial virus type a primer set:

SEQ ID NO. 7: forward primer (F): TGATACACTCAACAAAGATCAACTTCTG

SEQ ID NO. 8: reverse primer (R): TCTCCTGTGCTCCGTTGGAT

SEQ ID NO. 9: fluorescent probe (P): CATCCAGCAAATACAC, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

adenovirus primer set:

SEQ ID NO. 10: forward primer (F): ATGGCCACCCCTTCGATGA

SEQ ID NO. 11: reverse primer (R): CCCGGACTCAGGTACTCCGA

SEQ ID NO. 12: fluorescent probe (P): TACATGCACATCGCCGG, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

human metapneumovirus primer set:

SEQ ID NO. 13: forward primer (F): GCAACATGAAATAATGAAGAATGTACAC

SEQ ID No. 14: reverse primer (R): GAGTGCTAATGTTTTTACTCCTCGTTAA

SEQ ID NO. 15: fluorescent probe (P): TGAGCTCACACTATTGAA, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

respiratory syncytial virus type B primer set:

SEQ ID No. 16: forward primer (F): GCTTGATGTCAGTTGTGGAACAAT

SEQ ID NO. 17: reverse primer (R): TGTATCTCATTCAGCTTCGGTATGA

SEQ ID NO. 18: fluorescent probe (P): CACAAACATATGTCCTAATAGA, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

influenza a virus primer set:

SEQ ID NO. 19: forward primer (F): CTGGGAATGCTGAAATTGAAGATC

SEQ ID No. 20: reverse primer (R): TCATATCCACTGGCCACGGCAAG

SEQ ID NO. 21: fluorescent probe (P): CCTGAGAGGATCAGTGGCCCACAAGT, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

influenza b virus primer set:

SEQ ID NO. 22: forward primer (F): CCAGGGATTGCAGACATTGAAGA

SEQ ID NO. 23: reverse primer (R): TGGCTTCGTACCCAACCATAGAGTACT

SEQ ID No. 24: fluorescent probe (P): TCGTTGTTAGGCCCTCTGTGGCG, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

parainfluenza virus type 1 primer set:

SEQ ID No. 25: forward primer (F): ACCCGAAATGACAATTCC

SEQ ID NO. 26: reverse primer (R): GGATCATGATAATGAAGGACAA

SEQ ID NO. 27: fluorescent probe (P): TACACCAGCAGGAAGGACACA, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

parainfluenza virus type 2 primer set:

SEQ ID NO. 28: forward primer (F): CCAACTATCGATTTGCTG

SEQ ID NO. 29: reverse primer (R): ATCCGGTAGT ATTTAGTAGG

SEQ ID No. 30: fluorescent probe (P): AATGAGTCCAACCGAACCAATCC, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

parainfluenza virus type 3 primer set:

SEQ ID NO. 31: forward primer (F): ACCAGGAAACGATGAATG

SEQ ID NO. 32: reverse primer (R): GGGTT CACTCTCGATTTC

SEQ ID NO. 33: fluorescent probe (P): AGTATGACAGATGACACAATGCTCC, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

parainfluenza virus type 4 primer set:

SEQ ID No. 34: forward primer (F): GGTACAATTGCTCTTGGTG

SEQ ID NO. 35: reverse primer (R): ACTGCCTCATTTGTTTCTG

SEQ ID NO. 36: fluorescent probe (P): ACAAGTTACAGCAGCAATCGGTC, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

bocavirus primer set:

SEQ ID NO. 37: forward primer (F): GGAGAGGA AGAGACACTG

SEQ ID NO. 38: reverse primer (R): GGTGTTCCTGATGATATGAG

SEQ ID NO. 39: fluorescent probe (P): TCATCACAGGAGCAGGAGCC, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

2) cy5-BHQ2 fluorescence signal group

Legionella pneumophila primer set:

SEQ ID No. 40: forward primer (F): TCCTAGTAGTGAAGATGAATGTAATGATGT

SEQ ID NO. 41: reverse primer (R): AACTTAATATGAGATTTTCCCGCTGCCAACAA

SEQ ID No. 42: fluorescent probe (P): TGCTGCGGTACCCTGCGATTC, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

bordetella pertussis primer set:

SEQ ID No. 43: forward primer (F): GGCATCAAGCACCGCTTTAC-

SEQ ID NO. 44: reverse primer (R): CGGTGTTGGGAGTTCTG

SEQ ID No. 45: fluorescent probe (P): CGTGCCTGACTACTAGT, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

rhinovirus primer set:

SEQ ID No. 46: forward primer (F): AGACCAATAGTAGGCAAC

SEQ ID No. 47: reverse primer (R): CCTACTAAGCTCTGTGTA

SEQ ID No. 48: fluorescent probe (P): TCCAGGTTGTCTAAGGTCAAGCAC, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

enterovirus primer set:

SEQ ID NO. 49: forward primer (F): CGAACTCAGCATCAGAAG

SEQ ID No. 50: reverse primer (R): TCTCGTGTATGACATCCA

SEQ ID NO. 51: fluorescent probe (P): ACTACACCACCATCAACTATTACAACG, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

chlamydia pneumoniae primer set:

SEQ ID No. 52: forward primer (F): TGAAGTCGGAATTGCTAGTAATGG

SEQ ID No. 53: reverse primer (R): GTGTGTACAAGGCCCGAGAAC

SEQ ID No. 54: fluorescent probe (P): TGTCAGCCATAACGCCGTGAAT, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

mycoplasma pneumoniae primer set:

SEQ ID No. 55: forward primer (F): GGGAGGCAGCAGTAGGGAAT

SEQ ID NO. 56: reverse primer (R): ACAATCTTAAAGACCTTCATCGTTCA

SEQ ID No. 57: fluorescent probe (P): TTTTTCACAATGAGCGAAAGCTTGATGGA, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

coronavirus OC43 primer set:

SEQ ID No. 58: forward primer (F): GCCTATCA ACAACAAGATG

SEQ ID No. 59: reverse primer (R): GCAACACTTATATTATCATTTTCTC

SEQ ID No. 60: fluorescent probe (P): CTTATGACCACGCTGACGCTG, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

coronavirus NL63 primer set:

SEQ ID No. 61: forward primer (F): GTCGTTCTTCAACTCGTAA

SEQ ID No. 62: reverse primer (R): AAGCCAAAGTAACAGCAG

SEQ ID No. 63: fluorescent probe (P): CTTCTCGTAGCACTTCAAGACAACAG, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

primer set for coronavirus HKU 1:

SEQ ID NO. 64: forward primer (F): AGCTGACACTTCTACTCC

SEQ ID No. 65: reverse primer (R): GAACCTGGTCGACTATTAG

SEQ ID NO. 66: fluorescent probe (P): AGACCTTCCTGAGCCTTCAACAT, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

coronavirus C229E primer set:

SEQ ID NO. 67: forward primer (F): GTGGATTTGTCACCCAAG

SEQ ID No. 68: reverse primer (R): CCTGTAGGTTCAGTTTTAGC

SEQ ID NO. 69: fluorescent probe (P): CAGACGACACCTTCAACACGC, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

HIN1 primer set:

SEQ ID No. 70: forward primer (F): GTGGATTTGAGATGATTTG

SEQ ID NO. 71: reverse primer (R): GCAGTTCATTCCTGTTAA

SEQ ID No. 72: fluorescent probe (P): AACTTCCACTGTATCCTGACCAATCA, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

the simultaneous detection of SARS-CoV-2 virus and various pathogenic microorganisms is that after one nucleic acid extraction, the sample is subjected to two real-time fluorescence PCR detections to respectively determine SARS-CoV-2 virus and various pathogenic microorganisms in the sample.

Furthermore, the kit also comprises an internal quality control nucleic acid sample which is extracted as necessary, the internal quality control nucleic acid sample is used for simultaneously carrying out quality monitoring on SARS-CoV-2 virus sample detection and detection of a plurality of pathogenic microorganism samples, and the process of extracting the nucleic acid sample has no systematic error before the PCR reaction is determined.

The internal quality control nucleic acid sample is MS2 bacteriophage containing a section of HIV-1 target gene RNA fragment, and is added when the sample to be detected is subjected to nucleic acid extraction.

Preferably, when the plasma to be detected is pretreated, the plasma sample is split by proteinase K, and then the internal quality control nucleic acid sample is added.

Preferably, the nucleic acid of the sample is extracted and used

Extracting with a Virus Spin Kit nucleic acid extraction Kit.

Preferably, the kit further comprises an AC amplification quality control for detecting whether the multiple fluorescence channels can normally develop color, wherein the AC amplification quality control nucleic acid is added after the sample nucleic acid is extracted and before PCR amplification is carried out.

Preferably, the kit also comprises a positive nucleic acid fragment of each detection target virus as a positive quality control.

Preferably, the kit further comprises necessary PCR amplification reagents and fluorescence color development reagents.

The invention has the advantages that the invention can detect SARS-CoV-2 virus simply and rapidly, and can detect whether other pathogenic microorganisms exist in target plasma at the same time, thereby providing convenience for subsequent plasma inactivation treatment and blood product extraction.

Drawings

FIG. 1 shows the results of nucleic acid detection of SARS-CoV-2 virus in a plasma sample of sample No.3, wherein 1A is a negative control, 1B is a positive control, 1C is a blank control, and 1D is the result of nucleic acid detection (negative) of the sample.

Fig. 2 and sample No.3 show the results of multiplex pathogen detection in the plasma sample, 2A shows the negative control results of detection in the plurality of fluorescence channels, 2B shows the positive control results of detection in the plurality of fluorescence channels, and 2C shows the results of multiplex pathogen detection (negative) in the sample.

Detailed Description

Relates to a kit:

novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method)

Single-tube multiple respiratory tract pathogen nucleic acid detection kit (fluorescence PCR method)

Component name	Color of tube cap	Specification of
			RF reaction solution	Purple color	＞320μl
RF primer	White colour	＞450μl
			RF buffer 1	Red colour	＞1000μl
RF buffer 2	Blue color	＞1000μl
			RF reaction enzyme	Yellow colour	＞100μl
RF internal quality control	Black color	＞900μl
			RF positive quality control	Green colour	＞100μl
RF dilution	Is transparent	＞1500μl

Virus Spin Kit nucleic acid extraction Kit (57704)

Example 1 extraction of nucleic acid sample from plasma

1. Extracting total nucleic acid samples including RNA and DNA

Extracting a nucleic acid sample in blood plasma to be detected, wherein the nucleic acid extracted at one time is simultaneously used for amplifying novel coronavirus and detecting nucleic acid of respiratory pathogens, when the nucleic acid sample is extracted, preparation is prepared for the detection of SARS-CoV-2 and multiple respiratory pathogens in a second step, when the blood plasma nucleic acid is extracted, a necessary internal standard sample (an internal control product of a multiple respiratory pathogen nucleic acid detection kit) is added, and the specific operation steps are as follows:

1、

the Virus Spin Kit nucleic acid extraction Kit is combined with a single-tube multiple respiratory tract pathogen nucleic acid detection Kit to extract a plasma nucleic acid sample

(1) AW1, AW2, vector RNA and proteinase K were prepared as required in the kit.

(2) Mu.l of QIAGEN Protease were transferred into a 1.5ml microcentrifuge tube (Nasopharyngeal swab for comparison with respiratory tract sample Or the sputum sample, the plasma sample is more viscous and is not easy to crack, so the dosage of the proteinase K is increased from 25uL to 30uL)

(3) Add 200. mu.l of plasma or serum to a microcentrifuge tube (if the sample size is less than 200. mu.l, add an appropriate amount of 0.9% sodium chloride solution to bring the total protease and sample to 225. mu.l); this step was accompanied by the establishment of negative controls: 200 μ l of the dilution (from the pathogen kit) was used in the same manner as the samples.

(4) Adding 5 μ L of RF internal quality control (the RF internal quality control is MS2 bacteriophage containing a section of HIV-1 target gene RNA) to the plasma sample and the negative control; this step was accompanied by a negative control: adding 5 mul of RF internal quality control into 200 mul of diluent, and carrying out the same operation with the sample;

(5) add 200. mu.l Buffer AL (containing 28. mu.g/ml carrier RNA) and vortex mix for 30s with the lid;to ensure Efficient lysis, the sample and Buffer must be mixed AL is mixed well to produce a homogeneous solution, so here when lysis is properly extended Workshop。

(6) Incubate 15 minutes at 56 ℃ in a heat block;

(7) briefly centrifuge a 1.5ml tube to remove droplets inside the cap (a transient centrifuge may be used);

(8) to the sample was added 250. mu.l ethanol (96-100%), capped, and mixed thoroughly by vortex mixing for 30 s: (The vortex time is suitably prolonged so that the lysis is sufficient). Incubate lysate with ethanol for 5 minutes at room temperature (15-25 ℃);

(9) centrifuge the 1.5ml tube briefly to remove the droplets inside the cap;

(10) loading all lysate onto a QIAamp MinElute column, capping and centrifuging at 6000g for 1 minute, placing the QIAamp MinElute column in a clean 2ml collection tube and discarding the collection tube containing filtrate, and if the lysate does not pass completely through the column after centrifugation, centrifuging again at a higher speed until the QIAamp MinElute column is empty;

(11) open QIAamp MinElute column and add 600. mu.L of buffer AW1(Here, the elution with buffer is increased The volume is sufficient to elute the proteinase K, thereby avoiding the influence on the subsequent nucleic acid amplification) Centrifugation at 6000g for 1 minute placed the QIAampMinElute column in a clean 2ml collection tube and the collection tube containing the filtrate discarded;

(12) open QIAamp MinElute column and add 600. mu.L of buffer AW2(Here, the elution with buffer is increased The volume is sufficient to elute the proteinase K, thereby avoiding the influence on the subsequent nucleic acid amplification) Centrifuge at 6000g for 1 min. The QIAampMinElute column was placed in a clean 2ml collection tube and the collection tube containing the filtrate was discarded;

(13) open the QIAamp MinElute column and add 500. mu.l ethanol (96-100%) and centrifuge at 6000g for 1 min, discard the collection tube with filtrate;

(14) the QIAamp MinElute chromatography column was placed in a clean 2ml collection tube and centrifuged at 20,000g for 3 minutes to completely dry the membrane;

(15) the QIAamp MinElute chromatography column was placed in a new 2ml collection tube, the lid was opened, and then incubated at 56 ℃ for 3 minutes to completely dry the membrane;

(16) the QIAamp MinElute column was placed in a clean 1.5ml microcentrifuge tube, the lid of the QIAamp MinElute column was opened, and 30-50. mu.l of Buffer AVE was added to the center of the membrane (elution Buffer pre-equilibrated to room temperature, elution Buffer dispensed to the center of the membrane to completely elute bound RNA and DNA); incubated at room temperature for 1 minute, centrifuged at 20,000g for 1 minute,collecting centrifugate to obtain plasma nucleic acid sample containing internal quality control, and setting cathode in step (4) Nucleic acid sample obtained as a sexual control was used as a nucleic acid sample for the negative control in example 3. In this example, the nucleic acid sample obtained by final extraction, including RNA and DNA, is used as the nucleic acid template of the experimental sample.

The nucleic acid sample prepared in this example was then used for SARS-CoV-2 viral RNA detection in example 2 and multi-pathogen nucleic acid detection in example 3, respectively.

Example 2 detection of SARS-CoV-2 viral RNA

The Taqman fluorescent probe detection principle is used for detecting SARS-CoV-2 virus RNA,

the Taqman fluorescent probe is a section of specific oligonucleotide, and two ends of the specific oligonucleotide are respectively marked with a fluorescent group and a quenching group. When the probe is complete, the fluorescence emitted by the fluorescent group is absorbed by the quenching group; during PCR amplification, the probe is cut off by 5'→ 3' exonuclease activity of the polymerase, the fluorescence group and the quenching group are separated, fluorescence is emitted, the generation of each molecule of product is accompanied with the generation of one molecule of fluorescence signal, and the real-time monitoring of the whole PCR process is realized by monitoring the accumulation of the fluorescence signals.

Primers and probes:

SARS-CoV-2 target one (orf1ab):

forward primer (F): CCCTGTGGGTTTTACACTTAA

Reverse primer (R): ACGATTGTGCATCAGCTGA

Fluorescent probe (P): 5'-TEXAS RED610-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ2-3'

SARS-CoV-2 target two (N):

forward primer (F): GGGGAACTTCTCCTGCTAGAAT

Reverse primer (R): CAGACATTTTGCTCTCAAGCTG

Fluorescent probe (P): 5'-FAM-TTGCTGCTGCTTGACAGATT-BHQ1-3'

(1) Preparation of rRT-PCR reaction solution: taking out COV detection buffer solution D (containing a primer and a probe) and COV reaction solution (containing dNTP and enzyme) from the kit,

the following table was prepared for each reaction system tested:

components	Volume of
		COV detection buffer D	13μL
COV reaction solution	5μL
		Total amount of	18μL

(2) Preparation of blank control: without addition of any template, as a blank: (No requirement is made in the kit description The control is set up in practical operation and is used for monitoring whether the amplification system has cross contamination)；

Preparation of negative control: 1.5uLCOV negative quality control plus 0.5uL internal quality control (the internal quality control can be understood as amplification quality control, and amplification reaction monitoring is a section of plasmid containing human housekeeping gene sequence) is used as negative control;

preparation of positive control: the kit is provided with a positive quality control, the COV positive quality control is taken out from the kit, melted at room temperature, shaken and uniformly mixed, and then centrifuged for 10 seconds.

(3) Sample adding: mixing the sample nucleic acid (a)Nucleic acid samples prepared in example 1) Respectively taking 2 mu L of the COV positive quality control and the COV negative control, respectively adding the obtained product into a PCR tube, after sample adding, covering a tube cover, oscillating and uniformly mixing, and centrifuging for a short time;

(4) the following reaction program settings were followed:

(5) the baseline setting takes the fluorescent signal of 3-10 or 6-15 cycles, the threshold setting principle is that the threshold line just exceeds the highest point of the amplification curve of the negative control, and the threshold line can be adjusted according to the noise condition of the instrument.

(6) And (3) judging standard:

the blank control can not generate pathogen specific amplification and can not generate an internal quality control amplification curve;

the negative control can not generate pathogen specific amplification, and an internal quality control amplification curve is generated, if the negative control does not meet the internal quality control amplification curve, the PCR is prompted to have inhibitors or improper sample collection and storage, and repeated experiments are needed;

the positive control should show a pathogen-specific amplification curve, and if it does not match, the experiment should be repeated.

Example 3 multiplex pathogen nucleic acid detection

And (3) checking the principle: the respiratory tract pathogen detection kit is based on a single-tube multiplex PCR technology, and the technology can detect and analyze 13 target genes at the same time in one PCR. The kit adopts two-step PCR reaction,

(1) a first step of reverse transcription, performing a multiplex PCR reaction, the purpose of which is to enrich for target nucleic acids present in the sample;

(2) the first PCR product is then divided into two portions and two separate second PCR reactions are performed, which amplify pathogen specific regions, bind to pathogen probes, and analyze different pathogens by melting curves.

After the second PCR reaction, the system generates a melting curve by slowly raising the temperature and monitoring the change of the fluorescence signals of different fluorescence channels. The Tm values for the different fluorescence channels for a particular PCR product are constant and predictable. The pathogen species in different wells were statistically analyzed by analyzing the Tm values.

Primers and probes:

1 ROX to 6 target reaction

Target 1 (respiratory syncytial virus type a):

forward primer (F): TGATACACTCAACAAAGATCAACTTCTG

Reverse primer (R): TCTCCTGTGCTCCGTTGGAT

Fluorescent probe (P): 5'-ROX-CATCCAGCAAATACAC-BHQ1-3'

Target 2 (adenovirus):

forward primer (F): ATGGCCACCCCTTCGATGA

Reverse primer (R): CCCGGACTCAGGTACTCCGA

Fluorescent probe (P): 5'-ROX-TACATGCACATCGCCGG-BHQ1-3'

Target 3 (human metapneumovirus):

forward primer (F): GCAACATGAAATAATGAAGAATGTACAC

Reverse primer (R): GAGTGCTAATGTTTTTACTCCTCGTTAA

Fluorescent probe (P): 5 '-ROX-TGAGCTCACACTATTGAA-BHQ 1-3'

Target 4 (respiratory syncytial virus type B):

forward primer (F): GCTTGATGTCAGTTGTGGAACAAT

Reverse primer (R): TGTATCTCATTCAGCTTCGGTATGA

Fluorescent probe (P): 5'-ROX-CACAAACATATGTCCTAATAGA-BHQ1-3'

Target 5 (influenza a virus):

forward primer (F): CTGGGAATGCTGAAATTGAAGATC

Reverse primer (R): TCATATCCACTGGCCACGGCAAG

Fluorescent probe (P): ROX-CCTGAGAGGATCAGTGGCCCACAAGT-BHQl-3'

Target 6 (influenza b virus):

forward primer (F): CCAGGGATTGCAGACATTGAAGA

Reverse primer (R): TGGCTTCGTACCCAACCATAGAGTACT

Fluorescent probe (P): ROX-TCGTTGTTAGGCCCTCTGTGGCG-BHQl-3'

7-11 target reaction 1 Cy5

Target 7 (legionella pneumophila):

forward primer (F): TCCTAGTAGTGAAGATGAATGTAATGATGT

Reverse primer (R): AACTTAATATGAGATTTTCCCGCTGCCAACAA

Fluorescent probe (P): 5'-Cy5-TGCTGCGGTACCCTGCGATTC-BHQ2-3'

Target 8 (bordetella pertussis):

forward primer (F): GGCATCAAGCACCGCTTTAC-

Reverse primer (R): -CGGTGTTGGGAGTTCTG-

Fluorescent probe (P): 5'-Cy5-CGTGCCTGACTACTAGT-BHQ2-3'

Target 9 (rhinovirus):

forward primer (F): AGACCAATAGTAGGCAAC

Reverse primer (R): CCTACTAAGCTCTGTGTA

Fluorescent probe (P): 5'-Cy5-TCCAGGTTGTCTAAGGTCAAGCAC-BHQ2-3'

Target 10 (enterovirus):

forward primer (F): CGAACTCAGCATCAGAAG

Reverse primer (R): TCTCGTGTATGACATCCA

Fluorescent probe (P): 5'-Cy5-ACTACACCACCATCAACTATTACAACG-BHQ2-3'

Target 11 (chlamydia pneumoniae):

forward primer (F): TGAAGTCGGAATTGCTAGTAATGG

Reverse primer (R): GTGTGTACAAGGCCCGAGAAC

Fluorescent probe (P): cy5-TGTCAGCCATAACGCCGTGAAT-BHQ2-3'

Target 12 (mycoplasma pneumoniae):

forward primer (F): GGGAGGCAGCAGTAGGGAAT

Reverse primer (R): ACAATCTTAAAGACCTTCATCGTTCA

Fluorescent probe (P): cy5-TTTTTCACAATGAGCGAAAGCTTGATGGA-BHQ2-3'

13-17 target reaction 2 ROX

Target 13 (parainfluenza virus type 1):

forward primer (F): ACCCGAAATGACAATTCC

Reverse primer (R): GGATCATGATAATGAAGGACAA

Fluorescent probe (P): 5'-ROX-TACACCAGCAGGAAGGACACA-BHQ1-3'

Target 14 (parainfluenza virus type 2):

forward primer (F): CCAACTATCGATTTGCTG

Reverse primer (R): ATCCGGTAGT ATTTAGTAGG

Fluorescent probe (P): 5'-ROX-AATGAGTCCAACCGAACCAATCC-BHQ1-3'

Target 15 (parainfluenza virus type 3):

forward primer (F): ACCAGGAAACGATGAATG

Reverse primer (R): GGGTT CACTCTCGATTTC

Fluorescent probe (P): 5'-ROX-AGTATGACAGATGACACAATGCTCC-BHQ1-3'

Target 16 (parainfluenza virus type 4):

forward primer (F): GGTACAATTGCTCTTGGTG

Reverse primer (R): ACTGCCTCATTTGTTTCTG

Fluorescent probe (P): 5'-ROX-ACAAGTTACAGCAGCAATCGGTC-BHQ1-3'

Target 17 (bocavirus):

forward primer (F): GGAGAGGA AGAGACACTG

Reverse primer (R): GGTGTTCCTGATGATATGAG

Fluorescent probe (P): 5'-ROX-TCATCACAGGAGCAGGAGCC-BHQ1-3'

18-22 target reaction 2 Cy5

Target 18 (coronavirus OC43):

forward primer (F): GCCTATCA ACAACAAGATG

Reverse primer (R): GCAACACTTATATTATCATTTTCTC

Fluorescent probe (P): 5'-Cy5-CTTATGACCACGCTGACGCTG-BHQ2-3'

Target 19 (coronavirus NL63):

forward primer (F): GTCGTTCTTCAACTCGTAA

Reverse primer (R): AAGCCAAAGTAACAGCAG

Fluorescent probe (P): 5'-Cy5-CTTCTCGTAGCACTTCAAGACAACAG-BHQ2-3'

Target 20 (coronavirus HKU1):

forward primer (F): AGCTGACACTTCTACTCC

Reverse primer (R): GAACCTGGTCGACTATTAG

Fluorescent probe (P): 5'-Cy5-AGACCTTCCTGAGCCTTCAACAT-BHQ2-3'

Target 21 (coronavirus C229E):

forward primer (F): GTGGATTTGTCACCCAAG

Reverse primer (R): CCTGTAGGTTCAGTTTTAGC

Fluorescent probe (P): 5'-Cy5-CAGACGACACCTTCAACACGC-BHQ2-3'

Target 22(HIN1):

forward primer (F): GTGGATTTGAGATGATTTG

Reverse primer (R): GCAGTTCATTCCTGTTAA

Fluorescent probe (P): 5'-Cy5-AACTTCCACTGTATCCTGACCAATCA-BHQ2-3'

First step PCR reaction

(1) Preparing a PCR reaction system:

dissolving the RF reaction solution and the RF primer at 2-8 ℃, and adding 15 mu L of the solution into each PCR tube.

Component (tube cover color)	Volume of
		RF reaction liquid (purple)	6.25μl
RF primer (white)	8.75μl
		Total volume	15μl

Sample adding: add 10. mu.l of nucleic acid template to each PCR tube (positive and negative controls included)

Sample nucleic acid templates were the extracted test samples, negative controls and positive controls from the pathogen kit in example 1.

(2) Setting a first-step PCR reaction program:

(II) second step PCR reaction

(1) Preparation of PCR reaction System

(2) Taking out the PCR amplification product of the first step from the PCR instrument, standing for 5-10 min at 2-8 ℃, and then carrying out instantaneous centrifugation for 2min (so that the generation of aerosol can be effectively reduced, and cross contamination is avoided); taking 5 mu l of the reaction product of the first step PCR, and respectively adding the reaction product into the PCR tubes of the second step reaction system 1 and the reaction system 2;

(3) setting a second-step PCR reaction program:

detection channel

Detection channel	Fluorescent probe	Excitation wavelength	Emission wavelength
				FAM	BHQ1	465nm	510nm
ROX	ROX	465nm	610nm
				Cy5	Cy5	465nm	660nm

Second PCR temperature setting program

(III) interpretation of results

(1) Negative control interpretation criteria are given in the following table

Negative control Tm value

Negative samples: negative control as background, reaction system 1 or 2 had no specific peak in both channels of ROX and Cy5, and FAM channel had to have specific negative peak, and judged whether it was the internal quality control and amplification quality control AC (AC1 or AC2) negative peak according to Table 1. If there is a corresponding internal quality control and AC negative peak, the sample is a true negative sample. If internal quality control or AC negative peak is absent, the experimental result of the sample hole is invalid.

The internal quality control was 5. mu.l of the internal quality control added before extraction in example 1;

AC is amplification quality control, and whether the reaction systems 1 and 2 work or not is detected.

(2) Positive control interpretation criteria are given in the following Table

Positive control Tm value

Positive samples: as a background, reaction system 1 or 2 had specific peaks in both channels of ROX and Cy5, and the Tm values thereof were determined according to Table 3.

When the negative control and the positive control both meet the judgment standard, the analysis of the sample experiment result can be carried out; if the negative control or the positive control does not meet the judgment standard, the experiment is not established and needs to be detected again.

(3) Pathogen interpretation criteria are given in the following table

Tm value of pathogen

Example 4 methodological confirmation of the novel coronavirus nucleic acid detection System

According to the quality control requirement of plasma laboratory detection in the recovery period of the Xinguan pneumonia convalescent patient, the repeatability, the intermediate precision, the detection limit and the specificity of the method are confirmed, and the performance of at least more than 20 parts of plasma sample detection is confirmed.

1. Repeatability of

The purpose is as follows:

by detecting the internal quality control of the positive control and the negative control carried by the kit, whether the consistency of the positive control Ct value and the internal quality control Ct value among the results obtained by continuously detecting for multiple times under the same detection condition by the method is in accordance with the requirement is confirmed. The results are shown in the following table:

results of the repeatability test

2. Intermediate precision

The purpose is as follows:

and confirming whether the consistency of results obtained by continuously and repeatedly detecting the positive control Ct value and the internal quality control Ct value of the method under the same detection condition meets the requirement or not at different time by confirming different inspectors. The results are shown in the following table:

results of intermediate precision test

3. Detection limit

The purpose is as follows: the lowest standard substance concentration that can be detected by the method is evaluated. The results are shown in the following table:

test results of detection limit

Note: N/A represents that the Ct value of the detection result of the ABI7500 real-time fluorescent PCR instrument is underended. "+" indicates that the test result was positive, and "-" indicates that the test result was negative.

4. Specificity

4.1 crossover experiments

The purpose is as follows: additional pathogen material was added to both the known positive and known negative samples to confirm the specificity of the detection method.

The detection method and program:

53 respiratory tract infection related pathogen nucleic acids and human genome DNA are selected, added into SARS-CoV-2 positive and negative sample nucleic acids, and detected by using a novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method). Cross-pathogen nucleic acids were added to P0 and N0, and mixed positive samples were designated P1-P18 and mixed negative samples were designated N1-N18 according to pathogen group number.

53 pathogens associated with respiratory tract infection

The results are shown in the following table

Detection result of COV001N

Note: N/A represents that the Ct value of the detection result of the ABI7500 real-time fluorescent PCR instrument is underended. "+" indicates that the test result was positive, and "-" indicates that the test result was negative.

4.2 interference experiments

The purpose is as follows: an interfering substance is added to the known positive sample and the known negative sample to confirm the specificity of the detection method.

The detection method and program: 27 interfering substances are selected and added into COVID-19 positive and negative samples according to the experimental concentration, and after nucleic acid is extracted, the novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method) is used for detection. The positive samples were named P1-P27 and the negative samples were named N1-N27 according to the numbers of interfering substances.

27 interfering substances

Numbering	Interfering substances	Concentration of experiment	Numbering	Interfering substances	Concentration of experiment
						1	Mucins	1％v/v	15	Zanamivir	3mg/ml
2	Blood (human)	1％v/v	16	Ribavirin	1mg/ml
						3	Phenylephrine	10mg/ml	17	Oseltamivir	12mg/ml
4	Oxymetazoline	0.5mg/ml	18	Peramivir	3mg/ml
						5	Sodium chloride (with antiseptic)	0.9％	19	Lopinavir	200mg/ml
6	Beclomethasone	0.25mg/ml	20	Ritonavir	200mg/ml
						7	Dexamethasone	8mg/ml	21	Mupirocin	30mg/ml
8	Fluniprole	0.3mg/ml	22	Tobramycin	3mg/ml
						9	Triamcinolone acetonide	1.1mg/ml	23	Levofloxacin	150mg/ml
10	Budesonide	0.64mg/ml	24	Azithromycin	300mg/ml
						11	Mometasone	0.05％(g/g)	25	Cefepime	40mg/ml
12	Fluticasone	300mg/ml	26	Minocycline	70mg/ml
						13	Histamine hydrochloride	3mg/ml	27	Abidol	70mg/ml
14	Alpha-interferon	5MIU/ml

The results are shown in the following table:

detection result of COV002N

Note: N/A represents that the Ct value of the detection result of the ABI7500 real-time fluorescent PCR instrument is underended. "+" indicates that the test result was positive, and "-" indicates that the test result was negative.

5. Performance validation

The purpose is as follows: the method can be used for detecting SARS-CoV-2 nucleic acid in plasma of SARS-CoV-2 infected convalescent patient in the laboratory.

Results As shown in the following table, SARS-CoV-2 nucleic acid was not detected in the plasma of 20 convalescent patients:

results of Performance confirmation test

Example 5 methodological validation of the multiple pathogen detection System

According to the requirements of quality control of plasma laboratory detection in recovery period of convalescent coronary pneumonia patients, and by combining with the practical application condition of the method, the repeatability, intermediate precision, detection limit and specificity of the method are confirmed, and at least more than 20 parts of performance confirmation of plasma sample detection is carried out.

1. Repeatability of

The purpose is as follows:

by detecting the positive control and the negative control carried by the kit, whether the Tm values of the 4 pathogens of the positive control, the Tm value of the negative control, the internal quality control IC and the Tm values of the amplification quality control AC1 and AC2 meet the requirement of the consistency among the results of continuous multiple detection by the method under the same detection condition is confirmed. The results are shown in the following table:

positive control Tm value repeatability detection result

Negative control Tm value repeatability detection result

2. Intermediate precision

The purpose is as follows:

and (3) confirming whether the Tm values of 4 pathogens of the positive control, the Tm values of the negative control and the internal quality control IC and the Tm values of the amplification quality control AC1 and AC2 are consistent among results obtained by continuously and repeatedly detecting under the same detection condition by confirming different inspectors at different time.

Intermediate precision detection result of positive control Tm value

Intermediate precision detection result of negative control Tm value

3. Detection limit

The purpose is as follows: the lowest standard substance concentration that can be detected by the method is evaluated. Using digital PCR detection, the standard strain was diluted in a gradient up to the lowest detection limit and this concentration was taken for titre testing and digital PCR. The results of the measurements are shown in the following table.

Pathogens	Copy number/ml	Copy number/reaction
			Respiratory syncytial virus type A	558	19
Adenoviral vectors	45	2
			Human metapneumovirus	400	13
Respiratory syncytial virus type B	34	1
			Influenza A virus	413	14
Influenza B virus	160	5
			Bordetella pertussis	99	3
Rhinovirus	205	7
			Mycoplasma pneumoniae	678	23
Parainfluenza virus type 1	323	11
			Parainfluenza virus type 2	360	12
Parainfluenza virus type 3	837	28
			Bocavirus	480	16
Coronavirus OC43 type	349	12
			Coronavirus 229E type	266	9
Influenza A H1N1 virus	349	12

4. Specificity

4.1 specificity

The purpose is as follows: the specificity of the kit for detecting pathogens is determined. The kit is subjected to specificity verification by using the following seven pathogens, wherein the seven pathogens are respectively as follows: staphylococcus aureus, Streptococcus pneumoniae, beta hemolytic streptococcus, Klebsiella pneumoniae, herpes simplex virus, Coxsackie virus A, and Coxsackie virus B. These 7 pathogens were not in the detection range.

The results show that: none of these seven pathogens detected a positive signal.

4.2 interfering substances:

the purpose is as follows: to investigate the interference of the substances present in nasopharyngeal swabs or possibly introduced during sample handling with the detection, the present kit was tested using the following substances: human blood (containing) (1% v/v), human mucosal protein (1% v/v), human genome DNA (300ng), saline nasal spray (containing preservative) (1% v/v), and preservation solution.

The results show that: none of the above substances was found to affect the detection of the kit.

5. Performance validation

The purpose is as follows: the method can be used for detecting multiple pathogen nucleic acid in the plasma of SARS-CoV-2 infected convalescent patient in the laboratory.

Results as shown in the table below, no multiple pathogens were detected in the plasma of 20 convalescent patients.

Results of Performance confirmation test

"+" indicates that the test result was positive, and "-" indicates that the test result was negative.

Finally, it should be confirmed that the above embodiments are only used for helping those skilled in the art understand the essence of the present invention, and are not used for limiting the protection scope of the present invention.

SEQUENCE LISTING

<110> Wuhan blood products Limited, national drug group

<120> a method for simultaneously detecting SARS-CoV-2 virus and other pathogens in plasma of convalescent stage of convalescent coronary pneumonia patient

Method of producing a composite material

<160>72

<170>PatentIn version 3.5

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Claims (8)

1. A test kit for the simultaneous detection of SARS-CoV-2 virus and a plurality of other pathogens in a plasma sample, said kit comprising:

(1) a primer group for detecting SARS-CoV-2 virus,

(2) a detection primer group for detecting a plurality of other pathogens,

(3) necessary nucleic acid extraction reagent and color reagent;

the primer group for detecting SARS-CoV-2 virus is two groups of fluorescent PCR primer groups as follows:

SARS-CoV-2 target one (orf1ab) TEXAS RED610-BHQ2 fluorescence signal group

SEQ ID NO. 1: forward primer (F): CCCTGTGGGTTTTACACTTAA

SEQ ID NO. 2: reverse primer (R): ACGATTGTGCATCAGCTGA

SEQ ID NO. 3: fluorescent probe (P): CCGTCTGCGGTATGTGGAAAGGTTATGG, the 5 'end of the probe is TEXASRED610, and the 3' end is BHQ 2;

SARS-CoV-2 target two (N) FAM-BHQ1 fluorescence signal set

SEQ ID NO. 4: forward primer (F): GGGGAACTTCTCCTGCTAGAAT

SEQ ID No. 5: reverse primer (R): CAGACATTTTGCTCTCAAGCTG

SEQ ID NO. 6: fluorescent probe (P): TTGCTGCTGCTTGACAGATT, the 5 'end of the probe is FAM, and the 3' end is BHQ 1;

the primer group for detecting a plurality of other pathogens is

1) ROX-BHQ1 fluorescence signal set

Respiratory syncytial virus type a primer set:

SEQ ID NO. 7: forward primer (F): TGATACACTCAACAAAGATCAACTTCTG

SEQ ID NO. 8: reverse primer (R): TCTCCTGTGCTCCGTTGGAT

SEQ ID NO. 9: fluorescent probe (P): CATCCAGCAAATACAC, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

adenovirus primer set:

SEQ ID NO. 10: forward primer (F): ATGGCCACCCCTTCGATGA

SEQ ID NO. 11: reverse primer (R): CCCGGACTCAGGTACTCCGA

SEQ ID NO. 12: fluorescent probe (P): TACATGCACATCGCCGG, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

human metapneumovirus primer set:

SEQ ID NO. 13: forward primer (F): GCAACATGAAATAATGAAGAATGTACAC

SEQ ID No. 14: reverse primer (R): GAGTGCTAATGTTTTTACTCCTCGTTAA

SEQ ID NO. 15: fluorescent probe (P): TGAGCTCACACTATTGAA, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

respiratory syncytial virus type B primer set:

SEQ ID No. 16: forward primer (F): GCTTGATGTCAGTTGTGGAACAAT

SEQ ID NO. 17: reverse primer (R): TGTATCTCATTCAGCTTCGGTATGA

SEQ ID NO. 18: fluorescent probe (P): CACAAACATATGTCCTAATAGA, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

influenza a virus primer set:

SEQ ID NO. 19: forward primer (F): CTGGGAATGCTGAAATTGAAGATC

SEQ ID No. 20: reverse primer (R): TCATATCCACTGGCCACGGCAAG

SEQ ID NO. 21: fluorescent probe (P): CCTGAGAGGATCAGTGGCCCACAAGT, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

influenza b virus primer set:

SEQ ID NO. 22: forward primer (F): CCAGGGATTGCAGACATTGAAGA

SEQ ID NO. 23: reverse primer (R): TGGCTTCGTACCCAACCATAGAGTACT

SEQ ID No. 24: fluorescent probe (P): TCGTTGTTAGGCCCTCTGTGGCG, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

parainfluenza virus type 1 primer set:

SEQ ID No. 25: forward primer (F): ACCCGAAATGACAATTCC

SEQ ID NO. 26: reverse primer (R): GGATCATGATAATGAAGGACAA

SEQ ID NO. 27: fluorescent probe (P): TACACCAGCAGGAAGGACACA, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

parainfluenza virus type 2 primer set:

SEQ ID NO. 28: forward primer (F): CCAACTATCGATTTGCTG

SEQ ID NO. 29: reverse primer (R): ATCCGGTAGT ATTTAGTAGG

SEQ ID No. 30: fluorescent probe (P): AATGAGTCCAACCGAACCAATCC, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

parainfluenza virus type 3 primer set:

SEQ ID NO. 31: forward primer (F): ACCAGGAAACGATGAATG

SEQ ID NO. 32: reverse primer (R): GGGTT CACTCTCGATTTC

SEQ ID NO. 33: fluorescent probe (P): AGTATGACAGATGACACAATGCTCC, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

parainfluenza virus type 4 primer set:

SEQ ID No. 34: forward primer (F): GGTACAATTGCTCTTGGTG

SEQ ID NO. 35: reverse primer (R): ACTGCCTCATTTGTTTCTG

SEQ ID NO. 36: fluorescent probe (P): ACAAGTTACAGCAGCAATCGGTC, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

bocavirus primer set:

SEQ ID NO. 37: forward primer (F): GGAGAGGA AGAGACACTG

SEQ ID NO. 38: reverse primer (R): GGTGTTCCTGATGATATGAG

SEQ ID NO. 39: fluorescent probe (P): TCATCACAGGAGCAGGAGCC, the 5 'end of the probe is ROX, and the 3' end is BHQ 1;

2) cy5-BHQ2 fluorescence signal group

Legionella pneumophila primer set:

SEQ ID No. 40: forward primer (F): TCCTAGTAGTGAAGATGAATGTAATGATGT

SEQ ID NO. 41: reverse primer (R): AACTTAATATGAGATTTTCCCGCTGCCAACAA

SEQ ID No. 42: fluorescent probe (P): TGCTGCGGTACCCTGCGATTC, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

bordetella pertussis primer set:

SEQ ID No. 43: forward primer (F): GGCATCAAGCACCGCTTTAC-

SEQ ID NO. 44: reverse primer (R): CGGTGTTGGGAGTTCTG

SEQ ID No. 45: fluorescent probe (P): CGTGCCTGACTACTAGT, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

rhinovirus primer set:

SEQ ID No. 46: forward primer (F): AGACCAATAGTAGGCAAC

SEQ ID No. 47: reverse primer (R): CCTACTAAGCTCTGTGTA

SEQ ID No. 48: fluorescent probe (P): TCCAGGTTGTCTAAGGTCAAGCAC, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

enterovirus primer set:

SEQ ID NO. 49: forward primer (F): CGAACTCAGCATCAGAAG

SEQ ID No. 50: reverse primer (R): TCTCGTGTATGACATCCA

SEQ ID NO. 51: fluorescent probe (P): ACTACACCACCATCAACTATTACAACG, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

chlamydia pneumoniae primer set:

SEQ ID No. 52: forward primer (F): TGAAGTCGGAATTGCTAGTAATGG

SEQ ID No. 53: reverse primer (R): GTGTGTACAAGGCCCGAGAAC

SEQ ID No. 54: fluorescent probe (P): TGTCAGCCATAACGCCGTGAAT, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

mycoplasma pneumoniae primer set:

SEQ ID No. 55: forward primer (F): GGGAGGCAGCAGTAGGGAAT

SEQ ID NO. 56: reverse primer (R): ACAATCTTAAAGACCTTCATCGTTCA

SEQ ID No. 57: fluorescent probe (P): TTTTTCACAATGAGCGAAAGCTTGATGGA, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

coronavirus OC43 primer set:

SEQ ID No. 58: forward primer (F): GCCTATCA ACAACAAGATG

SEQ ID No. 59: reverse primer (R): GCAACACTTATATTATCATTTTCTC

SEQ ID No. 60: fluorescent probe (P): CTTATGACCACGCTGACGCTG, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

coronavirus NL63 primer set:

SEQ ID No. 61: forward primer (F): GTCGTTCTTCAACTCGTAA

SEQ ID No. 62: reverse primer (R): AAGCCAAAGTAACAGCAG

SEQ ID No. 63: fluorescent probe (P): CTTCTCGTAGCACTTCAAGACAACAG, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

primer set for coronavirus HKU 1:

SEQ ID NO. 64: forward primer (F): AGCTGACACTTCTACTCC

SEQ ID No. 65: reverse primer (R): GAACCTGGTCGACTATTAG

SEQ ID NO. 66: fluorescent probe (P): AGACCTTCCTGAGCCTTCAACAT, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

coronavirus C229E primer set:

SEQ ID NO. 67: forward primer (F): GTGGATTTGTCACCCAAG

SEQ ID No. 68: reverse primer (R): CCTGTAGGTTCAGTTTTAGC

SEQ ID NO. 69: fluorescent probe (P): CAGACGACACCTTCAACACGC, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

HIN1 primer set:

SEQ ID No. 70: forward primer (F): GTGGATTTGAGATGATTTG

SEQ ID NO. 71: reverse primer (R): GCAGTTCATTCCTGTTAA

SEQ ID No. 72: fluorescent probe (P): AACTTCCACTGTATCCTGACCAATCA, the 5 'end of the probe is Cy5, and the 3' end is BHQ 2;

the simultaneous detection of SARS-CoV-2 virus and various pathogenic microorganisms is that after one nucleic acid extraction, the sample is subjected to two real-time fluorescence PCR detections to respectively determine SARS-CoV-2 virus and various pathogenic microorganisms in the sample.

2. The kit of claim 1, wherein the kit further comprises,

necessary samples are extracted to obtain internal quality control nucleic acid samples, the internal quality control nucleic acid samples are used for simultaneously carrying out quality monitoring on SARS-CoV-2 virus sample detection and detection of a plurality of pathogenic microorganism samples, and the process of extracting the nucleic acid samples has no systematic error before confirming PCR reaction.

3. The kit of claim 1 or 2, wherein the internal quality control nucleic acid sample is MS2 bacteriophage containing a section of HIV-1 target gene RNA fragment.

4. The kit according to claim 1 or 2, wherein the internal quality control nucleic acid sample is added during nucleic acid extraction of a sample to be tested, preferably after the plasma sample is subjected to cleavage with proteinase K during pretreatment of the plasma to be tested.

5. The kit according to claim 1 or 2, wherein the nucleic acid is extracted from the sample

Extracting with a Virus Spin Kit nucleic acid extraction Kit.

6. The kit according to claim 1 or 2, further comprising,

and the AC amplification quality control is used for detecting whether the multiple fluorescence channels can normally develop color, and the AC amplification quality control nucleic acid is added after the sample nucleic acid is extracted and before PCR amplification is carried out.

7. The kit of claim 1 or 2, wherein the kit further comprises a positive nucleic acid fragment of each detected target virus as a positive quality control.

8. The kit of claim 1 or 2, wherein the kit further comprises necessary PCR amplification reagents, fluorescent color reagent.

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