CN112760414A - Primer set and application thereof - Google Patents
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Abstract
The invention provides a primer group and a primer group, wherein the primer group is used for S-L typing of SARS-CoV-2, and is characterized by comprising a first pair of primers and a first pair of probes, and the nucleic acid sequences of the primers are as follows: the first pair of primer upstream primer sequences: GTGGATGAGGCTGGTTCT (SEQ ID NO: 4); the downstream primer sequences of the first pair of primers: AACAACGCACTACAAGAC (SEQ ID NO: 5); first pair of probes first sequence: TCCTGTTCACCTTTTA (SEQ ID NO: 6); second sequence of first pair of probes: CAGCTTCCTGTTTACCT (SEQ ID NO: 7). The primer set provided by the invention can be used for efficiently and accurately typing SARS-CoV-2, and the typing result is used for guiding clinical treatment.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a primer group and application thereof, and more particularly relates to a kit, a SARS-CoV-2 detection method and a SARS-CoV-2 typing method.
Background
The outbreak of the new crown epidemic situation brings huge impact on global economy and health, and is key to the prevention, control, early detection, early isolation and early treatment of the new crown epidemic situation. Research has now shown that new coronaviruses can be divided into different types based on some single-site base mutation on the virus, with type S (28144 site C) being the earlier virus and type L (28144 site T) being evolutionarily later than type S. In addition, it has been found that D614(23403 site is A) and G614 (23403 site is G) are formed in the L-type virus, and a paper is published on-line in the journal of cell & lt 2 & gt, 7.7.2020 by the national laboratory of Ross and Alamous, USA, and D614G mutation is thought to increase the infectivity of the virus.
The current commonly used nucleic acid detection method RT-qPCR can only identify whether a new coronavirus is infected, but cannot identify the type of the virus. And when the type of the virus is judged, whole genome sequencing is required, and the method does not meet the requirement of rapid and wide screening in time period and cost.
Therefore, there is a need to develop an identification method that can be both identified and screened, and can be developed in most regions.
Disclosure of Invention
In the first aspect of the present invention, the present invention provides a primer set for S-L typing of SARS-CoV-2, comprising a first pair of primers and a first pair of probes, wherein the nucleic acid sequences of the primers are as follows: the first pair of primer upstream primer sequences: GTGGATGAGGCTGGTTCT (SEQ ID NO: 4); the downstream primer sequences of the first pair of primers: AACAACGCACTACAAGAC (SEQ ID NO: 5); first pair of probes first sequence: TCCTGTTCACCTTTTA (SEQ ID NO: 6); second sequence of the first pair of probes: CAGCTTCCTGTTTACCT (SEQ ID NO: 7). The first pair of primers and the first pair of probes according to the embodiment of the present invention are used for detecting 28144 th site of SARS-CoV-2 virus genome sequence (the sequence position of SARS-CoV-2 genome is referred to as NC-045512.2 sequence), the first sequence of the first pair of probes and the second sequence of the first pair of probes respectively carry different fluorophores, the first pair of primers can specifically amplify the sequence near the 28144 th site of SARS-CoV-2 virus genome sequence, when the 28144 th site is C, the first sequence of the first pair of probes can be complementarily paired with the virus genome sequence, when the nucleic acid guided by the first pair of primers is amplified to the position of the first sequence of the first pair of probes, the probes are broken and release the fluorophore which can be captured to the specificity of the first sequence of the first pair of probes, the 28144 th site is judged to be C by the captured fluorescence signal, further determining that the sample contains S-type virus of SARS-CoV-2; when the 28144 th site is T, the second sequence of the first pair of probes can be complementarily matched with the genome sequence of the virus, when the nucleic acid guided by the first pair of primers is amplified to the position of the second sequence of the first pair of probes, the probes are broken and release the captured fluorescent group specific to the second sequence of the first pair of probes, the 28144 th site is determined as T by the captured fluorescent signal, and the L-type virus containing SARS-CoV-2 in the sample is determined. According to the embodiment of the invention, the sample to be detected can be an environmental sample such as soil, water source and the like, a throat swab sample of an animal or human and the like.
According to an embodiment of the present invention, the primer set may further include at least one of the following additional features:
according to an embodiment of the invention, the nucleic acid sequences of the first pair of primers are as follows: the first pair of primer upstream primer sequences: ATCACCCATTCAGTACATCG (SEQ ID NO: 8); the downstream primer sequences of the first pair of primers: AACGCACTACAAGACTACCC (SEQ ID NO: 9). The inventors found that the first pair of primers can be sequences shown in SEQ ID NO 8 and SEQ ID NO 9, and that they can be used in combination with the first pair of probes to recognize and amplify 28144 th site and its vicinity in the SARS-CoV-2 virus genome sequence, thereby determining whether the SARS-CoV-2 virus is of L-type or S-type.
In the second aspect of the invention, the invention provides the primer group, the primer group is used for S-L typing of SARS-CoV-2, the primer group comprises a second pair of primers and a second pair of probes, and the nucleic acid sequences of the primers are as follows: the upstream primer sequence of the second pair of primers: CCAATTTAGGTTCCTGGCAATT (SEQ ID NO: 10); the downstream primer sequences of the second pair of primers: CACCCATTCAGTACATCGATATCG (SEQ ID NO: 11); second pair of probes first sequence: ATTGTAAAAGGTGAACAGG (SEQ ID NO: 12); second pair of probes second sequence: ATTGTAAAAGGTAAACAGG (SEQ ID NO: 13). The second pair of primers and the second pair of probes according to the embodiment of the invention are used for detecting the 28144 th site of SARS-CoV-2 virus genome sequence, the first sequence of the second pair of probes and the second sequence of the second pair of probes carry different fluorescent groups respectively, the second pair of primers can specifically amplify the sequence near the 28144 th site of SARS-CoV-2 virus genome sequence, when position 28144 is C, the first sequence of the second pair of probes can be complementarily paired with the genome sequence of the virus, when the nucleic acid guided by the second pair of primers is amplified to the position of the first sequence of the second pair of probes, the probes are broken off and the first sequence-specific fluorescent group of the second pair of probes which can be captured is released, judging that the 28144 th site is C through the captured fluorescence signal, and further judging that the sample contains S-type virus of SARS-CoV-2; when the 28144 th site is T, the second sequence of the second pair of probes can be complementarily matched with the genome sequence of the virus, when the nucleic acid guided by the second pair of primers is amplified to the position of the second sequence of the second pair of probes, the probes are broken off and release the captured second sequence-specific fluorescent group of the second pair of probes, the 28144 th site is T according to the captured fluorescent signal, and the L-type virus containing SARS-CoV-2 in the sample is judged.
In the third aspect of the invention, the invention provides a primer group, the primer group is used for D614G typing of SARS-CoV-2, and the SARS-CoV-2D614 and SARS-CoV-2G614 are both SARS-CoV-2L type. According to the embodiment of the invention, the primer set comprises a third pair of primers and a third pair of probes, and the nucleic acid sequences of the primers are as follows: third pair of primers first upstream primer sequence: ACCAGGAACAAATACTTC (SEQ ID NO: 29); third pair of primers first downstream primer sequence: GTAGGAGTAAGTTGATCTG (SEQ ID NO: 30); third pair of probes first sequence: TTCTTTATCAGG/lxna _ A/TGTTAACTGCAC (SEQ ID NO: 31); second sequence of third pair of probes: TTCTTTATCAGG/lxna _ G/TGTTAACTGCAC (SEQ ID NO: 32). According to the primer group of the embodiment of the invention, a locked nucleic acid technology is adopted, the 13 th position of the first sequence of the third pair of probes is an A base with a locked nucleic acid, the 13 th position of the second sequence of the third pair of probes is a G base with a locked nucleic acid, the first sequence of the third pair of probes and the second sequence of the third pair of probes are respectively provided with different fluorescent groups, when the 23403 site of SARS-CoV-2 virus is A, a fluorescent signal of the first sequence of the third pair of probes, or a fluorescent signal of the first sequence of the third pair of probes and a fluorescent signal of the second sequence of the third pair of probes can be detected by using an RT-qPCR technology; when the 23403 site of SARS-CoV-2 virus is G, only the fluorophore of the second sequence of the third pair of probes can be detected; if neither the fluorophore of the first sequence of the third pair of probes nor the fluorophore of the second sequence of the third pair of probes can be detected, new mutations of the virus at the site or abnormalities of the sample are considered, such as SARS-CoV-2 virus sample. The third pair of primers and the third pair of probes can be used alone for detecting SARS-CoV-2 virus and detecting and typing the 23403 locus of SARS-CoV-2 virus, or can be used together with the fourth group of primers and the fourth group of probes.
In the fourth aspect of the invention, the invention provides a primer group, the primer group is used for typing D614G of SARS-CoV-2, and the SARS-CoV-2D614 and SARS-CoV-2G614 are both of SARS-CoV-2L type. According to the embodiment of the present invention, the primer set comprises a fourth set of primers and a fourth set of probes, and the nucleic acid sequences of the primers are as follows: fourth set of primers first upstream sequence: ACCAGGAACAAATACTTC (SEQ ID NO: 29); fourth set of primers second upstream sequence: CTGCATGAATAGCAACAG (SEQ ID NO: 33); fourth set of primers first downstream sequence: GTAGGAGTAAGTTGATCTG (SEQ ID NO: 30); fourth group primer second downstream sequence: GACACTTGAGATTCTTGAC (SEQ ID NO: 34); fourth set of probes first sequence: TTCTTTATCAGG/lxna _ A/TGTTAACTGCAC (SEQ ID NO: 31); fourth set of probes second sequence: TGTGCAGTTAACA/lxna _ T/CCTGATAAAGAAC (SEQ ID NO: 35).
According to an embodiment of the present invention, the primer set may further include at least one of the following additional features:
according to an embodiment of the present invention, the second sequence nucleic acid sequence of the fourth set of probes is as follows: TGTGCAGTTAACA/lxna _ C/CCTGATAAAGAAC (SEQ ID NO: 36).
According to the specific embodiment of the present invention, the first sequence of the fourth group of probes is used in combination with the first upstream sequence of the fourth group of primers and the first downstream sequence of the fourth group of probes; the second sequence of the fourth group of probes is matched with the second upstream sequence of the fourth group of primers and the second downstream sequence of the fourth group of primers, wherein the second sequence of the fourth group of probes can be the sequence shown in SEQ ID NO. 35 or the sequence shown in SEQ ID NO. 36. And, the first sequence of the fourth group of probes is used in combination with the second sequence of the fourth group of probes. 31, the 13 th position of the fourth group probe first sequence is A base with locked nucleic acid, the 14 th position of the fourth group probe second sequence is T base with locked nucleic acid, the 14 th position of the fourth group probe second sequence is C base with locked nucleic acid, the fourth group probe first sequence and the fourth group probe second sequence have different fluorescent groups, when the fluorescent signal of the fourth group probe first sequence, or the fluorescent signal of the fourth group probe first sequence and the fluorescent signal of the fourth group probe second sequence are detected, the 23403 site of SARS-CoV-2 virus is A; when only the fluorescent signal of the second sequence of the fourth group probe is detected, the 23403 site of SARS-CoV-2 virus is indicated as G; when the fluorescence signal of the first sequence of the fourth group probe and any fluorescence signal of the second sequence of the fourth group probe can not be detected, the virus is proved to have new mutation at the site or the sample is abnormal, such as SARS-CoV-2 virus sample. The fourth group of primers and the fourth group of probes can be used alone for detecting SARS-CoV-2 virus and for detecting and typing 23403 locus of SARS-CoV-2 virus, or can be used together with the third pair of primers and the third pair of probes.
According to an embodiment of the present invention, the primer set in any one of the first to fourth aspects of the present invention further comprises: the nucleic acid sequences of the fifth pair of primers and the fifth probe are shown as follows: the sequence of the upstream primer of the fifth pair of primers is as follows: CAGGTATATGCGCTAGTTATCAGAC (SEQ ID NO: 1); the downstream primer sequences of the fifth pair of primers are as follows: CCAAGTGACATAGTGTAGGCAATG (SEQ ID NO: 2); fifth probe sequence: AGACTAATTCTCCTCGGCGGGCACG (SEQ ID NO: 3).
In a fifth aspect of the present invention, the present invention provides a primer set for use in the detection of SARS-CoV-2. According to an embodiment of the present invention, the primer set includes a fifth pair of primers and a fifth probe, and the nucleic acid sequences are as follows: the sequence of the upstream primer of the fifth pair of primers is as follows: CAGGTATATGCGCTAGTTATCAGAC (SEQ ID NO: 1); the downstream primer sequences of the fifth pair of primers are as follows: CCAAGTGACATAGTGTAGGCAATG (SEQ ID NO: 2); fifth probe sequence: AGACTAATTCTCCTCGGCGGGCACG (SEQ ID NO: 3). The fifth pair of primers and the fifth probe according to the embodiment of the invention are specific to the sequence of SEQ ID NO. 20 on the S gene of SARS-CoV-2, the sequence of SEQ ID NO. 20 is specific to SARS-CoV-2 virus, and the primer set can specifically recognize the sequence of SEQ ID NO. 20, thereby detecting SARS-CoV-2. The two ends of the fifth probe are respectively provided with a fluorescent group and a quenching group, the fluorescent group does not emit light at this time, the fifth probe can be complementarily paired with a part of the SEQ ID NO. 20 sequence, when the fifth pair of primers is subjected to synthesis amplification by taking the SEQ ID NO. 20 sequence as a template, the fifth probe is amplified to the position of the fifth probe, the fifth probe is broken to release the fluorescent group and the quenching group, the fluorescent group and the quenching group are far away at this time, the signal of the fluorescent group is captured, and a specific fluorescent signal is detected, which indicates that SARS-CoV-2 virus exists in a sample to be detected.
In a sixth aspect of the invention, the invention provides the use of an agent for the preparation of a kit for the detection of SARS-CoV-2, the agent comprising a primer set of any one of the sets provided in the first to fifth aspects of the invention. The reagent according to the embodiment of the invention can prepare a kit for efficiently and accurately detecting SARS-CoV-2 virus, and the prepared kit can efficiently and accurately detect whether a sample to be detected carries SARS-CoV-2 virus by using an RT-qPCR method, and can judge the type of the carried virus when any one group of primer sets from the first aspect to the fourth aspect of the invention is used, thereby effectively distinguishing S-L type virus and D614-G614 type virus of SARS-CoV-2.
In a seventh aspect of the invention, the invention provides the use of an agent for the preparation of a kit for typing SARS-CoV-2, the agent comprising any one of the sets of primer sets set forth in the first to fourth aspects of the invention. The reagent according to the embodiment of the invention can prepare the reagent kit for efficiently and accurately typing the SARS-CoV-2 virus, and the prepared reagent kit can efficiently and accurately utilize the RT-qPCR method to type the SARS-CoV-2 virus in a sample to be tested, and can effectively distinguish the S-type virus and the L-type virus of the SARS-CoV-2.
In an eighth aspect of the present invention, the present invention provides a kit for the detection of SARS-CoV-2. According to an embodiment of the present invention, the kit comprises any one of the primer sets set forth in the first to fifth aspects of the present invention. According to the kit provided by the embodiment of the invention, the primer group for detecting SARS-CoV-2 virus can be used for RT-qPCR detection, whether the sample to be detected carries SARS-CoV-2 virus or not is detected, after the sample to be detected carries SARS-CoV-2 virus, the primer group for typing SARS-CoV-2 virus can be used for typing SARS-CoV-2 virus, and the virus can be further distinguished into S type or L type, D614 type or G614 type.
In a ninth aspect of the invention, the invention provides a method for detecting SARS-CoV-2, which method is used for non-diagnostic purposes. According to the embodiment of the invention, the primer group provided by the fifth aspect of the invention is used for carrying out RT-qPCR detection on a sample to be detected, and the probe in the primer group provided by the fifth aspect of the invention further carries a fluorescent group; the detection of the fluorophore signal carried by the fifth probe is indicative of the presence of SARS-CoV-2 nucleic acid in the sample to be tested. According to the method of the embodiment of the invention, the fifth pair of primers and the fifth probe are specific to the sequence of SEQ ID NO. 20 on the S gene of SARS-CoV-2, the sequence of SEQ ID NO. 20 is specific to SARS-CoV-2 virus, and the primer group can specifically recognize the sequence of SEQ ID NO. 20, thereby detecting SARS-CoV-2. The two ends of the fifth probe are respectively provided with a fluorescent group and a quenching group, the fluorescent group does not emit light at the moment, the fifth probe can be matched with a part of the SEQ ID NO. 20 sequence in a complementary mode, when the fifth pair of primers is subjected to synthesis amplification by taking the part of the SEQ ID NO. 20 sequence as a template, the fifth probe is amplified to the position of the fifth probe, the fifth probe is broken off, the fluorescent group and the quenching group are released, the distance between the fluorescent group and the quenching group is far, the signal of the fluorescent group is captured, and a specific fluorescent signal is detected, so that SARS-CoV-2 virus exists in a sample to be detected.
CAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAG TCAATCCATCATTGCCTACACTATGTCACTTGG(SEQ ID NO:20)。
According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:
according to an embodiment of the invention, the fluorescent group is selected from at least one of the following: FAM, ROX, VIC, CY3, CY 5. The method according to the embodiment of the present invention may be used as long as it is a fluorophore that can be used for RT-qPCR.
In a tenth aspect of the invention, the invention provides a method for typing SARS-CoV-2, which method is used for non-diagnostic purposes. According to the embodiment of the invention, any one of the primer sets provided by the first aspect to the fourth aspect of the invention is used for performing RT-qPCR detection on a sample to be detected, and the probes in any one of the primer sets provided by the first aspect to the fourth aspect of the invention further carry fluorescent groups; detecting the fluorophore signal carried by the first sequence of the first pair of probes and/or the first sequence of the second pair of probes, which is indicative of SARS-CoV-2S type contained in the sample to be detected; detecting the fluorescent group signal carried by the second sequence of the first pair of probes and/or the second sequence of the second pair of probes, and indicating that the sample to be detected contains SARS-CoV-2L type; detecting the fluorescent group signal carried by the first sequence of the third pair of probes or/and detecting the fluorescent group signal carried by the first sequence of the fourth group of probes is an indication that the sample to be detected contains SARS-CoV-2D 614; the detection of the fluorophore signal carried by the second sequence of the third pair of probes and the absence of the fluorophore signal carried by the first sequence of the third pair of probes, or/and the detection of the fluorophore signal carried by the second sequence of the fourth set of probes and the absence of the first sequence of the fourth set of probes, is indicative of the presence of SARS-CoV-2G614 in the sample to be tested. According to the embodiment of the present invention, the first pair of primers and the first pair of probes and the second pair of primers and the second pair of probes are used for detecting the 28144 th site of SARS-CoV-2 virus genome sequence, the first sequence of the first pair of probes and the second sequence of the first pair of probes respectively carry different fluorophores, the first sequence of the second pair of probes and the second sequence of the second pair of probes respectively carry different fluorophores, the first pair of primers or the second pair of primers can specifically amplify sequences near the 28144 th site of SARS-CoV-2 virus genome sequence, when the 28144 th site is C, the first sequence of the first pair of probes or the first sequence of the second pair of probes can be complementarily paired with the virus genome sequence, when the nucleic acid guided by the first pair of primers is amplified to the position of the first sequence of the first pair of probes, or when the nucleic acid guided by the second pair of primers is amplified to the position of the first sequence of the second pair of probes, breaking the probe and releasing a captured fluorescent group, judging that the 28144 th site is C through a captured fluorescent signal, and further judging that the sample contains S-type virus of SARS-CoV-2; when the 28144 th site is T, the second sequence of the first pair of probes or the second sequence of the second pair of probes can be complementarily paired with the genome sequence of the virus, when the nucleic acid guided by the first pair of primers is amplified to the position of the second sequence of the first pair of probes or when the nucleic acid guided by the second pair of primers is amplified to the position of the second sequence of the second pair of probes, the probes are broken and release a fluorescent group which can be captured, the 28144 th site is determined to be T by a captured fluorescent signal, and the L-type virus containing SARS-CoV-2 in the sample is determined.
According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:
according to the embodiment of the invention, the first sequence of the third pair of probes and the second sequence of the third pair of probes respectively have different fluorophores, and when the 23403 site of SARS-CoV-2 virus is A, i.e. the virus is D614 type, the fluorescent signal of the first sequence of the third pair of probes, or the fluorescent signal of the first sequence of the third pair of probes and the fluorescent signal of the second sequence of the third pair of probes can be detected by using RT-qPCR technology; when the 23403 site of SARS-CoV-2 virus is G, i.e., the virus is G614 type, only the fluorophore of the second sequence of the third pair of probes can be detected; if neither the fluorophore of the first sequence of the third pair of probes nor the fluorophore of the second sequence of the third pair of probes can be detected, it is considered that the virus has a new mutation at the site or that the sample is abnormal, such as SARS-CoV-2 virus sample. The third pair of primers and the third pair of probes can be used alone for detecting SARS-CoV-2 virus and detecting and typing the 23403 locus of SARS-CoV-2 virus, or can be used together with the fourth group of primers and the fourth group of probes.
According to the specific embodiment of the present invention, the first sequence of the fourth group of probes is used in combination with the first upstream sequence of the fourth group of primers and the first downstream sequence of the fourth group of probes; the second sequence of the fourth group of probes is matched with the second upstream sequence of the fourth group of primers and the second downstream sequence of the fourth group of primers, wherein the second sequence of the fourth group of probes can be the sequence shown in SEQ ID NO. 35 or the sequence shown in SEQ ID NO. 36. And, the first sequence of the fourth group of probes is used in combination with the second sequence of the fourth group of probes. 31, the 13 th position of the fourth group probe first sequence is A base with locked nucleic acid, the 14 th position of the fourth group probe second sequence is T base with locked nucleic acid, the 14 th position of the fourth group probe second sequence is C base with locked nucleic acid, the fourth group probe first sequence and the fourth group probe second sequence have different fluorescent groups, when the fluorescent signal of the fourth group probe first sequence, or the fluorescent signal of the fourth group probe first sequence and the fluorescent signal of the fourth group probe second sequence are detected, the 23403 site of SARS-CoV-2 virus is A, namely the virus is D614 type; when only the fluorescent signal of the second sequence of the fourth group probe is detected, the 23403 site of SARS-CoV-2 virus is G, i.e. the virus is G614 type; when the fluorescent signal of the first sequence of the fourth group probe and any fluorescent signal of the second sequence of the fourth group probe can not be detected, the new mutation of the virus at the site or the abnormality of the sample can be shown, for example, the virus is not SARS-CoV-2 virus sample. The fourth group of primers and the fourth group of probes can be used alone for detecting SARS-CoV-2 virus and for detecting and typing 23403 locus of SARS-CoV-2 virus, or can be used together with the third pair of primers and the third pair of probes.
According to an embodiment of the invention, the fluorescent group is selected from at least one of the following: FAM, ROX, VIC, CY3, CY 5. The method according to the embodiment of the present invention may be used as long as it is a fluorophore that can be used for RT-qPCR.
In an eleventh aspect of the invention, the invention proposes the use of a kit according to the eighth aspect of the invention. According to an embodiment of the invention, the kit is used for guiding a fractionated treatment of a patient.
According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:
according to the embodiment of the invention, the sample to be detected shows positive fluorescence signal under the detection of the first sequence of the first pair of probes and/or the first sequence of the second pair of probes is an indication that the sample to be detected is from a patient with type S; and the second sequence of the first pair of probes and/or the second sequence of the second pair of probes in the sample to be detected shows a positive fluorescence signal under the detection, and the sample to be detected is an indication that the sample to be detected is from an L-shaped patient. According to an embodiment of the present invention, the S-type patient is a patient infected with SARS-CoV-2S-type virus, and the L-type patient is a patient infected with SARS-CoV-2L-type virus.
According to an embodiment of the invention, the clinical condition of the type S patient is more severe than that of the type L patient. Compared with L-type patients, the proportion of the S-type patients in heavy or dangerous weight is higher, and the probability of serious diseases of the S-type patients is higher.
Drawings
FIG. 1 is an amplification curve of 28144-PC-2 probe against pUC57-ORF8 according to an embodiment of the present invention;
FIG. 2 is an amplification curve of 28144-PC-2 probe against pUC57-ORF8-C according to an embodiment of the present invention;
FIG. 3 is an amplification curve of 28144-PC-3 probe against pUC57-ORF8 according to an embodiment of the present invention;
FIG. 4 is an amplification curve of 28144-PC-3 probe against pUC57-ORF8-C according to an embodiment of the present invention;
FIG. 5 is an amplification curve of 28144-PC-4 probe against pUC57-ORF8 according to an embodiment of the present invention;
FIG. 6 is an amplification curve of 28144-PC-4 probe against pUC57-ORF8-C according to an embodiment of the present invention;
FIG. 7 is an amplification curve of 28144-PC-5 probe against pUC57-ORF8 according to an embodiment of the present invention;
FIG. 8 is an amplification curve of 28144-PC-5 probe against pUC57-ORF8-C according to an embodiment of the present invention;
FIG. 9 is an amplification curve of 28144-PC-6 probe against pUC57-ORF8 according to an embodiment of the present invention;
FIG. 10 is an amplification curve of 28144-PC-6 probe against pUC57-ORF8-C according to an embodiment of the present invention;
FIG. 11 is an amplification curve of 28144-PC-4 probe against pUC57-ORF8-G according to an embodiment of the present invention;
FIG. 12 is an amplification curve of 28144-PC-4 probe against pUC57-ORF8-A according to an embodiment of the present invention;
FIG. 13 is an amplification curve of 28144-PC-5 probe against pUC57-ORF8-G according to an embodiment of the present invention;
FIG. 14 is an amplification curve of 28144-PC-5 probe against pUC57-ORF8-A according to an example of the present invention;
FIG. 15 is an amplification curve of 28144-PT-2 probe against pUC57-ORF8 according to an embodiment of the present invention;
FIG. 16 is an amplification curve of 28144-PT-2 probe against pUC57-ORF8-C according to an embodiment of the present invention;
FIG. 17 is an amplification curve of 28144-PT-3 probe against pUC57-ORF8 according to an embodiment of the present invention;
FIG. 18 is an amplification curve of 28144-PT-3 probe against pUC57-ORF8-C according to an embodiment of the present invention;
FIG. 19 is an amplification curve of 28144-PT-4 probe against pUC57-ORF8 according to an embodiment of the present invention;
FIG. 20 is an amplification curve of 28144-PT-4 probe against pUC57-ORF8-C according to an embodiment of the present invention;
FIG. 21 is an amplification curve of 28144-PT-5 probe against pUC57-ORF8 according to an embodiment of the present invention;
FIG. 22 is an amplification curve of 28144-PT-5 probe against pUC57-ORF8-C according to an embodiment of the present invention;
FIG. 23 is an amplification curve of 28144-PT-6 probe against pUC57-ORF8 according to an embodiment of the present invention;
FIG. 24 is an amplification curve of 28144-PT-6 probe against pUC57-ORF8-C according to an embodiment of the present invention;
FIG. 25 is an amplification curve of 28144-PT-7 probe against pUC57-ORF8 according to an embodiment of the present invention;
FIG. 26 is an amplification curve of 28144-PT-7 probe against pUC57-ORF8-C according to an embodiment of the present invention;
FIG. 27 is an amplification curve of 28144-PT-8 probe against pUC57-ORF8 according to an embodiment of the present invention;
FIG. 28 is an amplification curve of 28144-PT-8 probe against pUC57-ORF8-C according to an embodiment of the present invention;
FIG. 29 is an amplification curve of 28144-PT-7 probe against pUC57-ORF8-G according to an embodiment of the present invention;
FIG. 30 is an amplification curve of 28144-PT-7 probe against pUC57-ORF8-A according to an embodiment of the present invention;
FIG. 31 is an amplification curve of 28144-PT-8 probe against pUC57-ORF8-G according to an embodiment of the present invention;
FIG. 32 is an amplification curve of 28144-PT-8 probe against pUC57-ORF8-A according to an embodiment of the present invention;
FIG. 33 is an amplification curve of the LNA-A-P1 probe against pUC57-S in accordance with an embodiment of the invention;
FIG. 34 is an amplification curve of LNA-A-P1 against pUC57-S-G in accordance with an embodiment of the present invention;
FIG. 35 is an amplification curve of LNA-G-P1 against pUC57-S in accordance with an embodiment of the present invention;
FIG. 36 is an amplification curve of LNA-G-P1 against pUC57-S-G in accordance with an embodiment of the present invention;
FIG. 37 is an amplification curve of ROX-tagged LNA-A-P1 and VIC-tagged LNA-G-P1 against type D614 virus according to an embodiment of the invention;
FIG. 38 is an amplification curve at a viral load of 1000 copies/ml according to an embodiment of the invention;
FIG. 39 is an amplification curve of the LNA-A-P2 probe against pUC57-S in accordance with an embodiment of the present invention;
FIG. 40 is an amplification curve of the LNA-A-P2 probe against pUC57-S-G in accordance with an embodiment of the present invention;
FIG. 41 is an amplification curve of the LNA-G-P2 probe against pUC57-S according to an embodiment of the invention;
FIG. 42 is an amplification curve of the LNA-G-P2 probe against pUC57-S-G in accordance with an embodiment of the present invention;
FIG. 43 is an amplification curve for pUC57-S with a set of primer probes according to an embodiment of the present invention;
FIG. 44 is an amplification curve of a set of primer probes against pUC57-S-G according to an embodiment of the present invention;
FIG. 45 is an amplification curve for pUC57-S with a set of primer probes according to an embodiment of the present invention;
FIG. 46 is an amplification curve of a set of primer probes against pUC57-S-G according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Note that the nucleic acid site numbering information used in the present invention, for example, the 28144 site, was obtained from the SARS-CoV-2 genome (NC-045512.2) information published in the NCBI (national center for Biotechnology information) database.
The invention will be further explained with reference to specific examples. The experimental procedures used in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
First, the genome sequence of SARS-CoV-2 published so far was compared, and it was found that there was a sequence (SEQ ID NO:20) in the S gene contained only SARS-CoV-2 in the NCBI published so far, and a pair of primers S-F1 and S-R1 and a probe S-P1 were designed based on this, as shown in Table 1. The primer pair is used for detecting 5 samples which are detected to be positive by the current commercial kit and are SARS-CoV-2, and can be correctly identified, so the primer can be used as a primer for identifying SARS-CoV-2.
Table 1: primer design for identifying novel coronavirus
CAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCA ATCCATCATTGCCTACACTATGTCACTTGG(SEQ ID NO:20)
Example 2
In order to distinguish L type SARS-CoV-2 virus from S type SARS-CoV-2 virus, i.e. to distinguish 28144 locus, the invention designs multiple sets of primers, wherein the amplification primer pair includes F2/R2, F3/R3, F4/R4, and different probe primers need to be matched with different amplification primers, wherein the 28144-PT series probe sequence is aimed at the L type virus, i.e. 28144 locus is T, and a proper set of primers need to be found which can be correctly amplified and hybridized only when 28144 locus is T, thereby generating fluorescent signal, while when 28144 locus is C, G, A, no fluorescent signal can be generated. Similarly, the 28144-PC series probe sequence is directed against S-type virus, and also the 28144 site is C, and we need to find a proper set of primers which can correctly amplify and hybridize only when the 28144 site is C, so as to generate a fluorescent signal, but cannot generate a fluorescent signal when the 28144 site is T, G, A. The primer design is shown in table 2 below, and it should be noted that in this example, for the 28144-PT series probe sequence, a VIC fluorophore is selected, for the 28144-PC series probe sequence, an ROX fluorophore is selected, and all probes use MGB as a quencher, which is merely an example and can be replaced by other fluorophores or quenchers.
Table 2: primer and probe sequence for identifying S-type and L-type new coronavirus
We constructed 4 plasmids and cloned the ORF8 gene of SARS-CoV-2 containing the 28144 site to be identified on pUC57 as shown in Table 3 below:
table 3: plasmid information
| Name of plasmid | 28144 site base | Mimicking SARS-CoV-2 virus type |
| pUC57-ORF8 | T | L |
| pUC57-ORF8-C | C | S |
| pUC57-ORF8-A | A | No virus of this type has been reported |
| pUC57-ORF8-G | G | No virus of this type has been reported |
Firstly, the specificity of 28144-PC series probe sequences is tested by four plasmids shown in Table 3, namely 28144-PC-2, 28144-PC-3, 28144-PC-4, 28144-PC-6 and 28144-F2/R2 or 28144-F3/R3 are matched, and 28144-PC-5 and 28144-F4/R4 are matched, and the identification conditions of pUC57-ORF8 and pUC57-ORF8-C are tested. Attempts were made to select the reverse transcription fluorescence quantitative PCR enzymes (TAKARA and ROCHE) commonly used by two companies, and the results of the configuration of the transcription fluorescence quantitative PCR system and the reverse transcription fluorescence quantitative PCR reaction were shown in FIGS. 1 to 10 according to the system recommended by the specifications. FIG. 1 shows the result of detection of pUC57-ORF8 by 28144-PC-2 probe with 28144-F2/R2 primer, and FIG. 2 shows that amplification signals can be detected by 28144-PC-2 probe with 28144-F2/R2 primer and pUC57-ORF8-C, indicating that the 28144-PC-2 probe cannot distinguish pUC57-ORF8 from pUC57-ORF 8-C; FIG. 3 shows the result of detection of pUC57-ORF8 by 28144-PC-3 probe with 28144-F2/R2 primer, and FIG. 4 shows that amplification signals can be detected by 28144-PC-3 probe with 28144-F2/R2 primer and pUC57-ORF8-C, indicating that the 28144-PC-3 probe cannot distinguish pUC57-ORF8 from pUC57-ORF 8-C; FIG. 5 shows the result of detection of 28144-PC-4 probe with 28144-F2/R2 primer against pUC57-ORF8, FIG. 6 shows that 28144-PC-4 probe with 28144-F2/R2 primer against pUC57-ORF8-C, FIG. 5 shows that no amplification signal can be detected, FIG. 6 shows that an amplification signal can be detected, which indicates that 28144-PC-4 probe can distinguish pUC57-ORF8 from pUC57-ORF8-C and can detect pUC57-ORF8-C (i.e. SARS-CoV-2 type S); FIG. 7 shows the results of detection of 28144-PC-5 probe with 28144-F4/R4 primer against pUC57-ORF8, FIG. 8 shows 28144-PC-5 probe with 28144-F4/R4 primer against pUC57-ORF8-C, FIG. 7 shows that no amplification signal can be detected, FIG. 8 shows that an amplification signal can be detected, which indicates that 28144-PC-5 probe can distinguish pUC57-ORF8 from pUC57-ORF8-C and can detect pUC57-ORF8-C (i.e., SARS S-CoV-2); FIG. 9 shows the result of detection of pUC57-ORF8 by 28144-PC-6 probe with 28144-F2/R2 primer, and FIG. 10 shows that neither of the results of detection of pUC57-ORF8-C by 28144-PC-6 probe with 28144-F2/R2 primer shows that the 28144-PC-6 probe cannot distinguish pUC57-ORF8 from pUC57-ORF 8-C.
From the above results, 28144-PC-4 and 28144-PC-5 had good specificity for both L-type and S-type pseudoplasmids. Therefore, on the basis of this, 28144-PC-4 and 28144-PC-5 were used to test two other mock plasmids of mutant viruses which are not found but are possible, and the results are shown in FIGS. 11-14. FIG. 11 shows the result of detection of pUC57-ORF8-G by 28144-PC-4 probe with 28144-F2/R2 primer, and FIG. 12 shows that neither of the results of detection of pUC57-ORF8-A by 28144-PC-4 probe with 28144-F2/R2 primer indicates that pUC57-ORF8-G and pUC57-ORF8-A cannot be distinguished by 28144-PC-4 probe; FIG. 13 shows the result of detection of pUC57-ORF8-G by 28144-PC-5 probe with 28144-F4/R4 primer, and FIG. 14 shows that neither of the results of detection of pUC57-ORF8-A by 28144-PC-5 probe with 28144-F4/R4 primer indicates that the 28144-PC-5 probe can distinguish pUC57-ORF8-G from pUC57-ORF 8-A.
The results showed that 28144-PC-4 and 28144-PC-5 had specific fluorescent signals only against the S-type pseudoplasmid pUC57-ORF8-C, and thus could be used as primers for the identification of SARS-CoV-2 virus type S.
Next, the 28144-PT series probe sequences were tested for specificity, and the results are shown in FIGS. 15 to 28. FIG. 15 shows the result of detection of pUC57-ORF8 by 28144-PT-2 probe with 28144-F2/R2 primer, and FIG. 16 shows that amplification signals can be detected by 28144-PT-2 probe with 28144-F2/R2 primer and pUC57-ORF8-C, indicating that the 28144-PT-2 probe cannot distinguish pUC57-ORF8 from pUC57-ORF 8-C; FIG. 17 shows the result of detection of pUC57-ORF8 by 28144-PT-3 probe with 28144-F2/R2 primer, and FIG. 18 shows the result of detection of pUC57-ORF8-C by 28144-PT-3 probe with 28144-F2/R2 primer, both of which can detect amplification signals, indicating that the 28144-PT-3 probe cannot distinguish pUC57-ORF8 from pUC57-ORF 8-C; FIG. 19 shows the result of detection of pUC57-ORF8 by 28144-PT-4 probe with 28144-F2/R2 primer, and FIG. 20 shows that amplification signals can be detected by both results of detection of pUC57-ORF8-C by 28144-PT-4 probe with 28144-F2/R2 primer, indicating that the 28144-PT-4 probe cannot distinguish pUC57-ORF8 from pUC57-ORF 8-C; FIG. 21 shows the detection results of 28144-PT-5 probe with 28144-F2/R2 primer and pUC57-ORF8, FIG. 22 shows that 28144-PT-5 probe with 28144-F2/R2 primer and pUC57-ORF8-C, neither of the two results can detect amplification signals, which indicates that 28144-PT-5 probe cannot distinguish pUC57-ORF8 from pUC57-ORF 8-C; FIG. 23 shows the result of detection of 28144-PT-6 probe with 28144-F2/R2 primer against pUC57-ORF8, FIG. 24 shows that 28144-PT-6 probe with 28144-F2/R2 primer against pUC57-ORF8-C, neither of which can detect amplification signal, indicating that 28144-PT-6 probe cannot distinguish pUC57-ORF8 from pUC57-ORF 8-C; FIG. 25 shows the results of detection of 28144-PT-7 probe with 28144-F2/R2 primer, for pUC57-ORF8, FIG. 26 shows 28144-PT-7 probe with 28144-F2/R2 primer, for pUC57-ORF8-C, FIG. 25 shows that an amplification signal can be detected, FIG. 26 shows that an amplification signal cannot be detected, indicating that 28144-PT-7 probe can distinguish pUC57-ORF8 from pUC57-ORF8-C, and can detect pUC57-ORF8 (i.e., type L SARS-CoV-2); FIG. 27 shows the results of detection of 28144-PT-8 probe with 28144-F4/R4 primer, for pUC57-ORF8, FIG. 28 shows 28144-PT-8 probe with 28144-F4/R4 primer, for pUC57-ORF8-C, FIG. 27 shows that an amplification signal can be detected, FIG. 28 shows that an amplification signal cannot be detected, indicating that the 28144-PT-8 probe can distinguish pUC57-ORF8 from pUC57-ORF8-C, and can detect pUC57-ORF8 (i.e., type L SARS-CoV-2).
As can be seen from the above results, 28144-PT-7 and 28144-PT-8 have good specificity for both L-type and S-type pseudoplasmids. On the basis, 28144-PT-7 and 28144-PT-8 were used to test two other mimic plasmids of mutant viruses which are not found but are possible, and the results are shown in FIGS. 29-32. FIG. 29 shows the result of detection of pUC57-ORF8-G by 28144-PT-7 probe with 28144-F2/R2 primer, and FIG. 30 shows that neither of the results of detection of pUC57-ORF8-A by 28144-PT-7 probe with 28144-F2/R2 primer indicates that pUC57-ORF8-G and pUC57-ORF8-A can be distinguished by 28144-PT-7 probe; FIG. 31 shows the result of detection of pUC57-ORF8-G by 28144-PT-8 probe with 28144-F4/R4 primer, and FIG. 32 shows that neither of the results of detection of pUC57-ORF8-A by 28144-PT-8 probe with 28144-F4/R4 primer indicates that the 28144-PT-8 probe can distinguish pUC57-ORF8-G from pUC57-ORF 8-A.
The results showed that 28144-PT-7 and 28144-PT-8 only have specific fluorescent signals against the L-type pseudoplasmid pUC57-ORF8, and thus can be used as primers for identifying the L-type SARS-CoV-2 virus.
In conclusion, two pairs of primers capable of typing an L-type SARS-CoV-2 virus sample and an S-type SARS-CoV-2 virus sample are formed, wherein F2/R2 or F3/R3 is used as an amplification primer, and 28144-PC-4 and 28144-PT-7 are used as probes; and two sets of primers using F4/R4 as amplification primers 28144-PC-5 and 28144-PT-8 as probes.
Example 3
Generally, one kit can label up to 4 probes in order to be used in a wide range of areas. Therefore, 1 probe is set as 1 identification site, 2 probes are respectively the typing sites for detecting the L type SARS-CoV-2 virus and the S type SARS-CoV-2 virus, and 1 internal standard gene is used as collocation. Wherein the identification site can be a site recommended by each country at present, and a group of primers screened in example 1 or a group of primers for identifying ORF1ab site recommended by the Chinese CDC are selected as selectable identification primers. The typing primers were selected from one of the two sets of primers selected in example 2 for identifying L-type SARS-CoV-2 virus and S-type SARS-CoV-2 virus, and the internal standard primers were selected from the commonly used internal standard gene RP gene primers, wherein the ORF1ab site and the RP gene primers are shown in Table 4.
Table 4: primer sequence information of ORF1ab site and RP Gene
| Primer name | Primer sequence (5 '-3') |
| ORF1ab-F | CCCTGTGGGTTTTACACTTAA(SEQ ID NO:17) |
| ORF1ab-R | ACGATTGTGCATCAGCTGA(SEQ ID NO:18) |
| ORF1ab-P | CCGTCTGCGGTATGTGGAAAGGTTATGG(SEQ ID NO:19) |
| RP-F | AGATTTGGACCTGCGAGCG(SEQ ID NO:14) |
| RP-R | GAGCGGCTGTCTCCACAAGT(SEQ ID NO:15) |
| RP-P RNAse P | TTCTGACCTGAAGGCTCTGCGCG(SEQ ID NO:16) |
Based on the primer probe information shown in Table 4 and Table 2, 2 sets of combinations were performed, as shown in Table 5.
Table 5: primer and probe information in kit
It should be noted that the identification primer can be replaced with any currently recommended identification primer for SARS-CoV-2.
20 clinical samples in which 28144 site bases have been confirmed by one-generation sequencing (Sanger sequencing), namely, clinical samples in which typing of L-type SARS-CoV-2 or S-type SARS-CoV-2 has been confirmed, were selected for detection. The results of the tests carried out by the combination 1 and the combination 2 respectively show that the detection results of the kits of the combination 1 and the combination 2 are completely consistent with the results of the first-generation sequencing, which shows that the kit of the invention can be used for rapidly identifying SARS-CoV-2 and typing L-type SARS-CoV-2 or S-type SARS-CoV-2 of viruses.
Example 4 identification of viruses of D614 and G614 types
In order to identify SARS-CoV-2 virus types D614 and G614, i.e. to distinguish 23403 sites, the inventors designed sets of primers, wherein the amplification primer pair includes 23403F/23403R,23403F1/R1,23403F2/R2, and different probe primers need to be matched with different amplification primers, wherein the 23403-PA series probe sequence is directed against the virus type D614, i.e. 23403 site is A, and a proper set of primers need to be found only when 23403 site is A, so that the proper amplification and hybridization can be carried out, and a fluorescent signal can be generated. Similarly, the 23403-PG series probe sequence is directed against G614 type virus, also the 23403 site is G, and we need to find a proper set of primers only directed against 23403 site is G, which can be correctly amplified and hybridized, so as to generate fluorescent signal. The primer design is shown in table 6 below, and it should be noted that in this example, a VIC fluorophore is selected for an 23403-PA-series probe sequence, a ROX fluorophore is selected for a 23403 PG-series probe sequence, and MGB is used as a quencher for all probes, which is only an example and can be replaced by other fluorophores or quenchers.
Table 6: primer and probe sequence for identifying D614 and G614 new coronavirus
2 plasmids were constructed, and the S gene of SARS-CoV-2 containing the 23403 site to be identified was cloned in pUC57 as shown in Table 7 below:
table 7: plasmid information
| Name of plasmid | 23403 site base | Mimicking SARS-CoV-2 virus type |
| pUC57-S | A | D614 |
| pUC57-S-G | G | G614 |
The specificity of these primers was first tested with 4 plasmids as shown in Table 7. The reverse transcription fluorescence quantitative PCR enzyme (TAKARA company and ROCHE company) commonly used by two companies is tried to be selected and used for the configuration of the reverse transcription fluorescence quantitative PCR system and the reverse transcription fluorescence quantitative PCR reaction according to the system recommended by the specifications of the two companies, and the result shows that the primers can not show good specificity.
Therefore, two new sets of primers are used, and locked nucleic acid technology is used for probes of the two sets of primers. As shown in table 8:
table 8.: primer and probe sequence for identifying D614 and G614 new coronavirus
As a result of detecting pUC57-S with combination 3, LNA-A-P1 showed an amplification signal for pUC57-S, and LNA-A-P1 showed no amplification signal for pUC57-S-G, as shown in FIG. 34, as shown in FIG. 33. Therefore, LNA-A-P1 can be used as a primer for specific identification of D614, while LNA-G-P1 has an amplification signal against pUC57-S as shown in FIG. 35 and pUC57-S-G as shown in FIG. 36, and thus can be used as a universal identification probe. That is, when this probe primer is labeled with different fluorescent signals, such as ROX for LNA-A-P1 and VIC for LNA-G-P1, when the virus is of the D614 type, both ROX and VIC signals will be generated, as shown in FIG. 37. However, since VIC signal is weak compared to ROX signal, when viral load is low, only ROX signal may be generated, such as 1000 copies/ml viral load in fig. 38 generating only one ROX signal; when the virus is of the G614 type, only the universal probe primer signal, i.e., the VIC signal, is generated.
As a result of detecting pUC57-S with combination 4, LNA-A-P2 showed an amplification signal for pUC57-S, as shown in FIG. 39, and LNA-A-P2 showed an amplification signal for pUC57-S-G, as shown in FIG. 40. Therefore, LNA-A-P2 cannot be used as a primer for specific identification of D614G, but only as a universal identification primer. LNA-G-P2 showed an amplification signal for pUC57-S, FIG. 41, and pUC57-S-G also showed an amplification signal for 42, and thus was used as a universal identification primer and probe.
By using LNA-A-P2 as a general-purpose probe, and by using primers LNA-F2 and LNA-R2 in combination with specific probes LNA-A-P1 and primers LNA-F1 and LNA-R1 in combination 3, pUC57-S and pUC57-S-G were detected, respectively, and by detecting pUC57-S, the fluorescence signals of LNA-A-P1 and LNA-A-P2 were detected (FIG. 43), and when pUC57-S-G was detected, only the fluorescence signal of LNA-A-P2 was detected (FIG. 44).
By using LNA-G-P2 as a general-purpose probe, and by using primers LNA-F2 and LNA-R2 in combination with specific probes LNA-A-P1 and primers LNA-F1 and LNA-R1 in combination 3, pUC57-S and pUC57-S-G were detected, respectively, and by detecting pUC57-S, the fluorescence signals of LNA-A-P1 and LNA-G-P2 were detected (FIG. 45), and when pUC57-S-G was detected, only the fluorescence signal of LNA-G-P2 was detected (FIG. 46).
In conclusion, the primer probe sets of combination 4 and combination 3 in example 4 of the present invention can be used for typing and identifying the novel coronavirus of D614-G614.
Example 5
We retrospectively studied the nucleic acid samples of 271 patients with COVID-19 diagnosis in the A-zone. Treatment of COVID-19 patients investigated in this study allowed them to be in the region A confinement period, greatly limiting the spread of SARS-CoV-2 in the confined environment within region A. Therefore, these SARS-CoV-2 samples are well suited for investigating the differences in clinical characteristics associated with the S-and L-forms of SARS-CoV-2. We divided 271 patients into four groups, clinical severity, according to the guidelines for novel coronavirus diagnosis and treatment issued by the national institutes of health (7 th edition): asymptomatic (n-24), normal (n-98), severe (n-117) and critical (n-32). The proportion of patients with type S, who were either heavy or critically heavy, was significantly higher relative to patients with type L (P0.011, Fisher' S exact test, as shown in table 9). In addition, we classified asymptomatic and general patients into the "non-severe" category, and severe and critically ill patients into the "severe" category. The proportion of patients with type S (69.9%, 51/73) that belonged to the "severe" category was significantly higher compared to patients with type L (49.5%, 98/198) (P0.004, accurate Fisher test; as shown in table 9). To rule out the potential impact of age and underlying disease on the disease of patients of type S and L, we analyzed a subset of patients under 65 years of age without underlying disease (referred to as "group II"). It is noteworthy that in group II, when all four clinical severity levels were considered, the difference between patients with type S and L was not statistically significant, however, patients with type S were significantly more likely to belong to the "severe" category (P ═ 0.074, Fisher' S exact test, as shown in table 9). Thus, in group II patients, we observed a significantly higher proportion of patients with severe condition type S (61.5%, 16/26) than patients with type L (36.0%, 27/75) (P ═ 0.037, Fisher' S exact test, as shown in table 9). Therefore, after eliminating the underlying confounding factors (age and underlying disease), we have still found that patients with SARS-CoV-2 type are much more likely to develop severe disease than patients with type L. Overall, patients infected with SARS-CoV-2 type S are more clinically severe than patients with type L.
TABLE 9 correlation of SARS-CoV-2 typing with clinical features
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Wuhan Zhexi medical laboratory Co Ltd
<120> primer set and use thereof
<130> PIDC3204445
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<170> PatentIn version 3.5
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Claims (13)
1. A primer group for S-L typing of SARS-CoV-2, comprising a first pair of primers and a first pair of probes, wherein the nucleic acid sequences are as follows:
the first pair of primer upstream primer sequences: GTGGATGAGGCTGGTTCT (SEQ ID NO: 4);
the downstream primer sequences of the first pair of primers: AACAACGCACTACAAGAC (SEQ ID NO: 5);
first pair of probes first sequence: TCCTGTTCACCTTTTA (SEQ ID NO: 6);
second sequence of first pair of probes: CAGCTTCCTGTTTACCT (SEQ ID NO: 7).
2. The primer set of claim 1, wherein the nucleic acid sequence of the first pair of primers is as follows:
the first pair of primer upstream primer sequences: ATCACCCATTCAGTACATCG (SEQ ID NO: 8);
the downstream primer sequences of the first pair of primers: AACGCACTACAAGACTACCC (SEQ ID NO: 9).
3. A primer group, which is used for S-L typing of SARS-CoV-2, comprises a second pair of primers and a second pair of probes, and the nucleic acid sequences are shown as follows:
the upstream primer sequence of the second pair of primers: CCAATTTAGGTTCCTGGCAATT (SEQ ID NO: 10);
the downstream primer sequences of the second pair of primers: CACCCATTCAGTACATCGATATCG (SEQ ID NO: 11);
second pair of probes first sequence: ATTGTAAAAGGTGAACAGG (SEQ ID NO: 12);
second pair of probes second sequence: ATTGTAAAAGGTAAACAGG (SEQ ID NO: 13).
4. A primer set for typing SARS-CoV-2D 614G, wherein the SARS-CoV-2D614 and SARS-CoV-2G614 are both of the SARS-CoV-2L type, and the primer set comprises a third pair of primers and a third pair of probes, and the nucleic acid sequences are as follows:
the third pair of primer upstream primer sequences: ACCAGGAACAAATACTTC (SEQ ID NO: 29);
the downstream primer sequences of the third pair of primers are as follows: GTAGGAGTAAGTTGATCTG (SEQ ID NO: 30);
third pair of probes first sequence: TTCTTTATCAGG/lxna _ A/TGTTAACTGCAC (SEQ ID NO: 31);
second sequence of third pair of probes: TTCTTTATCAGG/lxna _ G/TGTTAACTGCAC (SEQ ID NO: 32).
5. A primer set, the primer set is used for D614G typing of SARS-CoV-2, the SARS-CoV-2D614 and SARS-CoV-2G614 are both the SARS-CoV-2L type, and the primer set comprises a fourth group of primers and a fourth group of probes, the nucleic acid sequences are shown as follows:
fourth set of primers first upstream sequence: ACCAGGAACAAATACTTC (SEQ ID NO: 29);
fourth set of primers second upstream sequence: CTGCATGAATAGCAACAG (SEQ ID NO: 33);
fourth set of primers first downstream sequence: GTAGGAGTAAGTTGATCTG (SEQ ID NO: 30);
fourth group primer second downstream sequence: GACACTTGAGATTCTTGAC (SEQ ID NO: 34);
fourth set of probes first sequence: TTCTTTATCAGG/lxna _ A/TGTTAACTGCAC (SEQ ID NO: 31);
fourth set of probes second sequence: TGTGCAGTTAACA/lxna _ T/CCTGATAAAGAAC (SEQ ID NO: 35);
optionally, the fourth set of probes has a second sequence nucleic acid sequence as shown below:
TGTGCAGTTAACA/lxna_C/CCTGATAAAGAAC(SEQ ID NO:36)。
6. the primer set according to any one of claims 1 to 5, further comprising a fifth primer pair and a fifth probe, wherein the nucleic acid sequences of the fifth primer pair and the fifth probe are as follows:
the sequence of the upstream primer of the fifth pair of primers is as follows: CAGGTATATGCGCTAGTTATCAGAC (SEQ ID NO: 1);
the downstream primer sequences of the fifth pair of primers are as follows: CCAAGTGACATAGTGTAGGCAATG (SEQ ID NO: 2);
fifth probe sequence: AGACTAATTCTCCTCGGCGGGCACG (SEQ ID NO: 3).
7. A primer group for detecting SARS-CoV-2, comprising a fifth pair of primers and a fifth probe, wherein the nucleic acid sequences are as follows:
the sequence of the upstream primer of the fifth pair of primers is as follows: CAGGTATATGCGCTAGTTATCAGAC (SEQ ID NO: 1);
the downstream primer sequences of the fifth pair of primers are as follows: CCAAGTGACATAGTGTAGGCAATG (SEQ ID NO: 2);
fifth probe sequence: AGACTAATTCTCCTCGGCGGGCACG (SEQ ID NO: 3).
8. Use of reagents for the preparation of a kit for the detection of SARS-CoV-2, characterized in that the reagents comprise a primer set according to any of claims 1 to 7.
9. Use of a reagent for the preparation of a kit for typing SARS-CoV-2, wherein the reagent comprises a primer set according to any one of claims 1 to 5.
10. A kit for detecting SARS-CoV-2, comprising the primer set according to any one of claims 1 to 5 or claim 7.
11. A method for detecting SARS-CoV-2, which method is for a non-diagnostic purpose,
performing RT-qPCR detection on a sample to be detected by using the primer set of any one of claims 1 to 7, wherein the probe in the primer set of any one of claims 1 to 7 further carries a fluorescent group;
detecting the fluorophore signal carried by any one of the probes 1-7, which is indicative of SARS-CoV-2 nucleic acid contained in the sample to be tested;
optionally, the fluorophore is selected from at least one of: FAM, ROX, VIC, CY5, CY 3.
12. A method for typing SARS-CoV-2, which is used for non-diagnostic purposes, characterized in that the primer set of any one of claims 1 to 5 is used to perform RT-qPCR detection on a sample to be tested, and the probe in the primer set of any one of claims 1 to 5 further carries a fluorescent group;
detecting the fluorophore signal carried by the first sequence of the first pair of probes and/or the first sequence of the second pair of probes, which is indicative of SARS-CoV-2S type contained in the sample to be detected;
detecting the fluorophore signal carried by the second sequence of the first pair of probes and/or the second sequence of the second pair of probes, which is indicative of SARS-CoV-2L contained in the sample to be detected;
detecting the fluorescent group signal carried by the first sequence of the third pair of probes or/and detecting the fluorescent group signal carried by the first sequence of the fourth group of probes is an indication that the sample to be detected contains SARS-CoV-2D 614;
the detection of the fluorophore signal carried by the second sequence of the third pair of probes and the non-detection of the fluorophore signal carried by the first sequence of the third pair of probes, or/and the detection of the fluorophore signal carried by the second sequence of the fourth set of probes and the non-detection of the first sequence of the fourth set of probes is indicative that the sample to be tested contains SARS-CoV-2G 614;
optionally, the fluorophore is selected from at least one of: FAM, ROX, VIC, CY5, CY 3.
13. Use of a kit according to claim 10 for guiding a patient in a fractionated treatment;
optionally, the sample to be tested shows a positive fluorescence signal under the detection of the first sequence of the first pair of probes and/or the first sequence of the second pair of probes is an indication that the sample to be tested is from a type S patient;
the sample to be detected shows a positive fluorescence signal under the detection of the second sequence of the first pair of probes and/or the second sequence of the second pair of probes, and the sample to be detected is an indication that the sample to be detected is from an L-shaped patient;
optionally, the clinical condition of the patient is more severe in the patient with S type than in the patient with L type, which transmits more strongly than S type.
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| CN116912783A (en) * | 2023-09-14 | 2023-10-20 | 苏州中科苏净生物技术有限公司 | State monitoring method and system of nucleic acid detection platform |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113278733A (en) * | 2021-05-21 | 2021-08-20 | 广东粤港澳大湾区国家纳米科技创新研究院 | Combination of primer and probe for detecting mutant strain of new coronavirus |
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| CN114107572A (en) * | 2022-01-26 | 2022-03-01 | 潮州凯普生物化学有限公司 | Primer probe set for detecting different new coronavirus mutant strains based on multiplex PCR technology, detection kit and application of detection kit |
| CN114107572B (en) * | 2022-01-26 | 2022-04-12 | 潮州凯普生物化学有限公司 | Primer probe set for detecting different new coronavirus mutant strains based on multiplex PCR technology, detection kit and application of detection kit |
| CN116912783A (en) * | 2023-09-14 | 2023-10-20 | 苏州中科苏净生物技术有限公司 | State monitoring method and system of nucleic acid detection platform |
| CN116912783B (en) * | 2023-09-14 | 2023-12-26 | 苏州中科苏净生物技术有限公司 | State monitoring method and system of nucleic acid detection platform |
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