PROTEASE ASSAYS AND THEIR APPLICATIONS

Abstract

The application describes methods for detecting site specific proteases indicative of infection by a protease-generating pathogen. The application also describes fusion proteins for use in the methods, DNAs encoding the proteins and cells that express them. Particular applications are described including fusion proteins and methods for detecting corona viruses,.

such as SARS CoV2. Method for protease and pathogen detection described in the application include protease amplification methods and methods using inhibitors to increase sensitivity and specificity.

Claims

1. A method for detection of a protease activity indicative of an infection by a pathogen in a host from which the sample is collected, the method comprising: a. Contacting the sample with a reaction mixture containing a light signal enabling molecule modified with an amino acid sequence that can be specifically cleaved with a protease being detected and other components and conditions sufficient to enable light signal production; b. Contacting a negative control sample with the same reaction mixture as in a, c. Optionally contacting the sample with a second reaction mixture containing a specific inhibitor for the protease; d. Incubating a and b for a certain period of time; e. Measuring the signal in a and b; f. Determining whether the protease activity is present in the sample by comparing signal change between a and b, wherein a change in signal intensity above a cutoff value indicates an infection by the pathogen; g. Or determining whether the protease activity is present in the sample by comparing signal change between a and c, wherein a change in signal intensity above a cutoff value indicates an infection by the pathogen.

2. The method of claim 1, wherein specific cleavage of the signal enabling molecule by the protease leads to change in signal intensity as compared to that of negative control, thereby indicating an infection of the pathogen.

3. The method of claim 1, wherein specific inhibition of the protease activity leads to change in signal intensity as compared to that without an inhibitor, thereby indicating an infection of the pathogen.

4. The method of claim 1, wherein the pathogen to be detected is human immunodeficiency virus (HIV), human hepatitis virus type C (HCV), coronavirus (CoV), dengue virus (DENV), West Nile virus (WNV), Zika virus, or any other virus encoding a viral protease able to cleave a specific amino acid sequence.

5. The method of claim 4, wherein the protease is any one or more of the HIV retropepsin protease (also known as the HIV retroviral aspartyl protease), the HCV NS3/NS4 serine protease, the coronavirus chymotrypsin-like (3CL) protease or papain, the dengue virus NS2/NS3 protease, the West Nile Virus NS2/NB3 protease, and the Zika virus NS2B/NS3 protease;

6. The method of claim 1, wherein the protease specific amino acid sequence is inserted within the light signal enabling molecule, wherein the insertion does not cause significant loss of the light signal enabling activity of the molecule.

7. The method of claim 6, wherein cleavage of the protease specific amino acid sequence in the signal enabling molecule by protease in a sample causes loss or decrease in the light signal intensity compared to a negative control sample, thereby indicating a pathogen infection in the host.

8. The method of claim 1, wherein the protease specific amino acid sequence is comprised in a linker sequence fused to the N or C terminus of the light signal enabling molecule on one end and to an inactivating moiety on the other end, whereby the light signal enabling molecule is inactivated.

9. The method of claim 8, wherein cleavage of the linker by the protease leads to recovery of the activity of light enabling molecule;

10. The method of claim 9, wherein increase of light signal in a reaction indicates the presence of protease in the sample and thereby indicates a pathogen infection in the host.

11. The method of claim 1, wherein the signal enabling molecule is linked to removable entity through a linker containing a cleavage site of a protease indicative of a pathogen infection.

12. The method of claim 11, wherein the removable entity can be removed from the reaction along with the signal enabling molecule unless the linker is cleaved by the protease in a sample.

13. The method of claim 11, wherein the loss of activity of the signal enabling molecule in the reaction is indicative of the presence of protease in the sample and thereby indicate a pathogen infection of the host.

14. The method of claim 1, wherein the light signal enabling molecule is a luciferase, a peroxidase, or an alkaline phosphatase.

15. The method of claim 14, wherein the luciferase is a firefly luciferase, a click beetle luciferase or a bacterial luciferase

16. The method of claim 1, wherein the protease specific amino acid sequence comprises a sequence of at least three amino acids.

17. The method of claim 16, wherein the protease specific amino acid sequence is flanked by additional amino acids not needed for recognition by the protease.

18. The method of claim 1, wherein the pathogen is a coronavirus.

19. The method of claim 18, wherein the coronavirus proteases used for coronavirus infection detection are the coronavirus papain protease and/or 3CL protease.

20. A method for detection of a protease activity indicative of an infection by a pathogen in a host from which the sample is collected, comprising. a. Contacting a sample with a reaction mixture containing a recombinant fusion protein of a signal enabling molecule linked to a protease through a linkage with an amino acid sequence that can be specifically cleaved by a protease being detected; b. Contacting a negative control sample with the same reaction mixture as in a, c. Optionally contacting the sample with a second reaction mixture containing a specific inhibitor of the protease; d. After incubation for a certain period of time, measuring the signal in a and b; e. Comparing signal change between a and b, wherein a change in signal intensity above a cutoff value indicates the presence of the protease and an infection by the pathogen; f. Or determining whether the protease activity is present in the sample by comparing the signals measured for a and c, wherein a change in the signal intensity above a cutoff value indicates the presence of the protease and an infection by the pathogen.

21. The method of claim 20, wherein the intact fusion protein has very little or no signal enabling or protease activity and cleavage of the linkage by a protease in the sample activates both the signal enabling molecule and protease from the fusion protein.

22. The method of claim 21, wherein the activated protease from the fusion protein in turn cleaves more fusion protein and activating both more signal enabling molecules and more protease, thus amplifying the signal generated by the signal enabling molecule.

23. The method of claim 20, wherein the signal enabling molecule is a light signal enabling molecule, a fluorescence enabling molecule, or an enzyme that produces a detectable product detectable product.

24. The method of claim 20, wherein the protease is encoded by a pathogen gene and can cleave a specific amino acid sequence, whereby the protease activity is indicative of an infection by the pathogen.

25. A method for detection of SARS COV-2 infection, the method comprising: a. Detection of a sample with a coronavirus protease assay first and, if positive, b. Detection of the sample with a second test specific for SARS CoV-2 infection or a second sample is collected from the same individual and tested with a second test specific for SARS CoV-2.

26. The method of claim 25, wherein the coronavirus protease is the papain like protease or 3CL.

27. The method of claim 25, wherein the second test specific for SARS CoV-2 infection is a RT-PCR based assay using specific primers or an antigen assay using specific antibodies.

28. A fusion protein comprising: (1) a first region of a signal producing polypeptide; (2) a second region of the signal producing polypeptide and (3) a linker polypeptide that connects (1) and (2) and comprises a cleavage site for a site-specific protease, wherein the signal producing polypeptide is active in the intact fusion protein and is inactivated when the cleavage site is cut by a protease.

29. A DNA encoding the fusion protein according to claim 28.

30. A cell comprising a DNA according to claim 29.

31. A fusion protein comprising: (1) a signal producing polypeptide; (2) a blocking polypeptide that inactivates the signal producing polypeptide in the fusion protein and (3) a linker polypeptide that connects (1) and (2) and comprises a cleavage site for a site specific protease, wherein the signal producing polypeptide is activated when the linker is cut by a protease.

32. A DNA encoding the fusion protein according to claim 31

33. A cell comprising a DNA according to claim 32

34. A fusion protein comprising: (1) a signal producing polypeptide; (2) a site specific protease polypeptide and (3) a linker polypeptide that connects (1) and (2) and comprises a cleavage site for the site specific protease, whereby the signal producing polypeptide and the protease polypeptide both are inactive when they are connected by the linker in the fusion protein, and both are activated when the linker is cut by the protease.

35. A fusion protein according to claim 34, further comprising an additional linker polypeptide at the end of the protease polypeptide distal to linker polypeptide connecting the signal producing polypeptide to the protease polypeptide.

36. A DNA encoding the fusion protein according to claim 34.

37. A cell comprising a DNA according to claim 36.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is a diagram depicting an embodiment in which a decrease in signal indicates the presence of a protease. A light signal enabling molecule [1] comprises moiety 1 [2] and moiety 2 [3] that together with one another to produce the light signal [ Moieties [2] and [3] are linked together by a linker [4] containing an amino acid sequence containing the cleavage site (amino acid sequence) of a protease [6], which does not cause significant loss of the light signally activity of the molecule. In the absence of the protease the light signal enabling molecule is active in producing a light signal [5]. In the presence of a protease that acts on the cleavage site, the linker is cut, moieties 2 and 3 are separated and the light signal enabling molecule is inactivated. The resulting reduction of the light signal is indicative of the presence of the protease activity that acts on the cleavage site. This, in turn, is indicative of the presence of infection of a pathogen that engenders the productions of the protease.

[0029] FIG. 2 is a diagram depicting an embodiment in which an increase in light is indicative of the presence of a protease. Light signal enabling molecule [1] is inactive when it is linked to an inactivating entity [7] through linker [4], which contains the cleavage site (amino acid sequence) for a protease [6]. (Other numbers are the same as for FIG. 1.) In the absence of a protease the acts on the cleavage site, the light signal enabling molecule is inactive and does not produce a light signal. In the presence of a protease that acts on the cleavage site, the linker is cleaved by the protease, resulting in dissociation of the inactivating moiety, and activation of the light signal enabling molecule. The resulting light signal and/or increased light signal is indicative of the presence of a protease that can cleave the linker, and this in turn is indicative of the presence of infection with the pathogen that engenders production of the protease.

[0030] FIG. 3 is a diagram depicting an embodiment in which an increase in light production indicates the presence of a protease and, thereby, the presence of a pathogen. A light signal enabling molecule [1] is attached to a removable entity [8] through a linker [4] that contains a cleavage site (amino acid sequence) for protease [6] and by itself does not cause loss of activity of the light signal enabling molecule. (Other numbers are as in preceding Figures.) When the light signal enabling molecule is attached to removable entity is can be removed from the reaction. When the linker is cleaved by the protease being detected,. the removable entity is dissociated from the light signal enabling molecule. The dissociated light signal enabling molecule will remain in the reaction when the removable entity is removed. The resulting retention of the light signal, compared to a negative control, indicates the presence of the protease and therefore the pathogen infection. One type of removable entity is a magnetic particle, which can be removed through the use of a magnet.

[0031] FIG. 4 is a diagram depicting the detection principle of one embodiment. In this embodiment, a protease inhibitor [9] specific for the protease being detected is used in the assay. (Other numbers are the same as in the preceding Figures.) The assay is conducted in two reactions, which are identical except that one contains the inhibitor or inhibitors. If the presence of the inhibitor or inhibitors leads to a detectable change in light signal, then the sample contains the protease being detected. Signal change can be increase or decrease of the signal. The diagram depicts a design where inhibition of the protease by the inhibitor leads to increase of the signal.

[0032] FIG. 5 is a diagram depicting an embodiment utilizing protease enabled signal amplification. A signal enabling molecule [1] is linked through a linker [4] comprising a protease cleavage site (amino acid sequence) to a protease [6]. When linked together both the signal enabling molecule and the protease are inactivated, producing little or no detectable signal. However, in the presence of protease that acts on the cleavage site, the linker is cut, releasing and activating both the signal enabling molecule and the protease. The protease thus released itself then cleaves the linker producing more signal and activating still more protease, leading to amplification of the signal. Because the protease is catalytic the amplification will be exponential.

[0033] An example of this type of embodiment is a fusion protein comprising a signal enabling protein, such as luciferase connected by a protease cleavable sequence to a viral protease, wherein the luciferase and the protease are inactive in the fusion, and regain their activity when the linker is cut.

[0034] FIG. 6 is a photograph of SDS-PAGE gel of recombinant mLuc protein digested with a recombinant 3CL enzyme. mLuc is a recombinant firefly luciferase containing a 3CL cleavage sequence.

[0035] FIG. 7 is a histogram showing the relative activity (RA) of positive controls (P1 and P2), negative control (N), and purified COVID-19 virus in serial dilutions. None of the purified virus dilution showed any 3CL activity.

[0036] FIG. 8 is a diagram depicting a fusion protein used in an embodiment of a PESA based assay. The fusion protein comprises a signal generating polypeptide region, [1], a first linker polypeptide region [4], a protease, and an optional second linker polypeptide region [10]. The first and the second linker (if present) comprise a protease cleavage site. The signal generating polypeptide is inactive when it is connected to the first linker polypeptide region in the fusion protein, and is active when it is released from the fusion protein, as by cleavage at the protease cleavage site in the linker. The protease likewise is inactive when is connected to the first linker polypeptide region in the fusion protein, active when is released from the fusion protein, as by cleavage at the protease cleavage site in the linker. The active protease released from the fusion protein recognizes and cleaves the proteases cleavage sites in the linkers in the fusion protein releasing additional active signal generating polypeptide and active protease, in a self amplifying reaction. The second linker polypeptide region decreases as much as 100% any residual activity of the protease in the fusion protein, should there be any. Additional linker with additional cleavage sites is present in additional embodiments in this regard Likewise, additional protease and signal generating entities can be incorporated into fusion proteins useful in this aspect of embodiments of inventions herein described.

DESCRIPTION

[0037] Herein described are protease assays for research and for clinical diagnosis of an infection of a pathogen. In some embodiments the assays depend on two factors to enable specific and sensitive detection of a pathogen or an infection caused by a pathogen: a pathogen encoded protease capable of only cleaving an amino acid sequence with specific characteristics, and a light signal enabling molecule.

[0038] Many pathogens, particularly viruses, produce specific proteases, which can cleave only an amino acid sequence with specific characteristics such as amino acid sequences. The genome of human immunodeficiency virus (HIV), for example, encodes a retropep sin, which cleaves specific sequence in the HIV polyprotein. The genome of HCV encodes a NS3/NS4 serine protease that cleaves four specific sites in the HCV polyproteins.

[0039] Specific inhibitors had been identified for these proteases and used as effective antiviral medicines.

[0040] Many other viruses also have specific proteases encoded by the viral genomes. Additional examples include, but are not limited to: coronavirus, whose genomes encode encode a papain-like (PL) protease and a 3-chymotrypsin-like (3CL) protease; the dengue virus, whose genome encodes the NS2/NS3 protease; the West Nile Virus, whose genome encodes the NS2/NB3 protease; and the Zika virus, whose genome encodes the NS2B/NS3 protease.

[0041] Intense efforts are being directed to developing inhibitors targeting these proteases with the intention that will be useful as antiviral medicines.

[0042] In spite of the development of highly successful antivirals targeting these proteases, these pathogen proteases have not been used for diagnosis purposes. There are several advantages of using specific proteases for diagnosis of a pathogen infection. Pathogen proteases appear early in the infection. This means pathogen proteins, including proteases, can be detected before pathogen specific antibodies can be detected. When properly designed, detection of an enzyme activity can be more sensitive than detection of a non-enzyme protein, e.g., an antigen using a pair of antibodies. In addition, because these proteases are unique and specific for their target cleavage amino acid sequences, a homogeneous assay can be designed so that there would be no need for washing, which simplifies the assay and required instrument. Moreover, since the protease activity is essential for the life cycle of a pathogen, it is less susceptible to genetic changes, a frequent problem associated with detection of pathogens, particularly viruses. The fact that pathogen proteases are not commonly used in diagnosis of pathogen infection is primarily due to lack of sensitivity of commonly used protease assays.

[0043] Assays disclosed herein overcome these limitations. Assays are described that use light enabling molecule to generate chemiluminescence or biochemiluminescence, resulting in high sensitivity. Light enabling molecules in accordance therefore generally should be a protein or otherwise contain an amino acid sequence, that can be modified by insertion of a protease cleavage site.

[0044] In some embodiments, the light enabling molecule is a molecule enabling biochemiluminescence. One requirement of the light enabling molecule is that it can be modified to contain an amino acid sequence with the protease cleavage site without resulting in significant loss of activity. In some embodiments, cleavage of the modified molecule by the protease leads to loss of light enabling activity. In other embodiments, cleavage of the modified molecule does not lead to loss of activity, but rather, leads to loss of the capability of being physically removed from the reaction.

[0045] An example of a light signal enabling molecule is a luciferase. Many species of insects and bacteria produce luciferase to generate light, which is believed to be a mating signal in the dark. One example of insect luciferases is the firefly luciferase. Firefly luciferase can be split into two complementary moieties that can still generate light when they are in close proximity, even though they are separate entities and they lose their light-generating activity when they are separated from one another. The two moieties can be held together by a linking amino acid sequence containing a protease cleavage site without causing loss of luciferase activity. Cleavage at the cleavage site by a protease allows the two moieties to separate from one another resulting in loss of the luciferase activity.

[0046] Firefly luciferase can also be modified at its N- or C-terminus without causing loss of activity. For example, streptavidin has been fused to the N- or C-terminus of firefly luciferase. These fusion proteins normally contain extra more flexible amino acid sequence between the fused protein and luciferase. Thus, an amino acid sequence containing the protease cleavage site can be inserted between the fusion protein and luciferase. In some embodiments described herein a streptavidin-protease cleavage site-luciferase fusion protein is used for detection of the protease activity in the sample. In this assay format, the luciferase activity can be removed from the reaction using biotinylated magnetic particles unless the protease cleavage site is cleaved by the protease activity in the sample. At least one negative control is assayed along with the sample.

[0047] In an embodiment, the sample or negative control is incubated with the streptavidin-protease cleavage site-luciferase fusion protein for a period of time, followed by incubation with biotinylated magnetic particles. After removal of the magnetic particles, the remaining solution is assayed to detect luciferase activity (by adding a solution containing appropriate concentrations of ATP, DTT, CoA, Magnesium salt and luciferin). Increase in light signal in the sample as compared to that in the negative control indicates the presence of protease activity in the sample, which in turn indicates an infection of the host from whom the sample is collected.

[0048] Many potent and specific inhibitors of pathogen proteases have been developed as therapeutic drugs or drug candidates. Because a drug or drug candidate is normally highly specific for a protease of a pathogen, it can be used for specific detection of a protease or to improve the specificity of a protease assay. In an assay where a specific protease inhibitor is used, the assay is carried out in two reactions, one of which contains one or more protease inhibitor. If the protease activity is inhibited as indicated by the light signal change, then the protease is present in the sample.

[0049] In some embodiments, detection of protease activity in a sample uses a signal amplification method, which depends on the protease activity being detected, to achieve even higher sensitivity. For convenience, this signal amplification method is termed “protease enabled signal amplification”, or PESA. This technology is called PESA technology. An assay based on the PESA technology is called PESA based assay.

[0050] The PESA technology is best understood by referring to FIG. 5. In one embodiment, a signal enabling molecule [1] is linked in a fusion protein with a protease [7]. The linkage between the signal enabling molecule and the protease contains a specific cleavage site for a protease. When physically linked together in the fusion protein both the signal enabling molecule and protease are inactive. When a protease cleaves the fusion protein at the specific cleavage site in the linkage between the signal enabling molecule and protease both are freed from the fusion protein and thus activated. The activated protease thus liberated cleaves the recombinant fusion protein, freeing and activating additional protease and signal enabling molecule in a self-perpetuating cycle, thus amplifying the signal. All the reactions are enzymatic resulting in substantial, in some cases exponential, signal amplification.

[0051] The signal enabling molecule in the fusion for a PESA-based assay can be those enabling chemiluminescence or biochemiluminescence as described above. It can also be an entity that produces a product that can be detected by other means. Examples of these signal enabling molecules include, but are not limited to, RNA polymerases such as T7 RNA polymerase, enzymes that can convert ADP or AMP to ATP, sequence specific nucleases, kinases, nonspecific nucleases such as exonucleases, and yet another proteases.

[0052] A variety of PESA based assays can be designed.

[0053] In some embodiment, the fusion protein is a fusion between the signal enabling molecule at the N terminus and the protease at the C terminus, linked by a linker that contains a protease cleavage site. To lower the possibility of autocleavage by the protease in the fusion protein, another protease cleavage site can be introduced to the C terminus of the protease in the fusion. In still other embodiment, both the N terminus and C terminus of the fusion contain additional protease cleavage sites so that both N and C termini are flanked by additional sequences to further minimize background activity from autocleavage. An example of PESA based assay is provided in Example 4.

[0054] Embodiments of inventions herein described include a variety of fusion proteins.

[0055] One embodiment in this regard is a fusion protein comprising: (1) a first region of a signal producing polypeptide; (2) a second region of the signal producing polypeptide and (3) a linker polypeptide that connects (1) and (2) and comprises a cleavage site for a site-specific protease,

[0056] wherein the signal producing polypeptide is active in the intact fusion protein and is inactivated when the cleavage site is cut by a protease.

[0057] Another embodiment in this regard is fusion protein comprising: (1) a signal producing polypeptide; (2) a blocking polypeptide that inactivates the signal producing polypeptide in the fusion protein and (3) a linker polypeptide that connects (1) and (2) and comprises a cleavage site for a site specific protease,

[0058] wherein the signal producing polypeptide is activated when the linker is cut by a protease.

[0059] Another embodiment in this regard is a fusion protein comprising: (1) a signal producing polypeptide; (2) a site specific protease polypeptide and (3) a linker polypeptide that connects (1) and (2) and comprises a cleavage site for a site specific protease, whereby the signal producing polypeptide and the protease polypeptide both are inactive when they are connected by the linker in the fusion, and both are activated when the linker is cut by a protease.

[0060] This embodiment is a signal amplification construct. When the initial linker cleavage by a protease releases not only the signal producing polypeptide but also releases the protease which in turn cleaves the linkers in other copies of the fusion protein releasing yet more signal producing polypeptide and protease, resulting in a multiplicative amplification of the signal.

[0061] In various related embodiments, the signal producing polypeptide and/or the protease can be flanked by additional linkers in the fusion protein, to facilitate their release and activation.

[0062] Additional embodiments in these regards provide polynucleotides encoding the fusion proteins described above, including unmodified and modified RNA and DNA. Such embodiments include cloning vectors, including plasmid, bacteriophage and viral vectors of all kinds, which are known to the art.

[0063] Embodiments include cells comprising the aforementioned polynucleotides encoding fusion proteins, particularly cells for producing the fusion proteins.

ILLUSTRATIVE EXAMPLES

[0064] The Examples below are illustrative of various aspects and embodiments of inventions herein disclosed but are in no ways limitative thereof. A complete understanding the inventions in this application is to have only by reading the entirety of the disclosure, including the claims, of the application and those of priority documents, in the context of the prior art as a whole, with the understanding of a person of skill in the art.

Example 1: Firefly Luciferase as the Light Signal Enabling Molecue for Detection of Protease Activity of a Coronavirus

[0065] In this example, firefly luciferase is used as the light signal enabling molecule for detection of the protease activity of a coronavirus. In this biochemiluminescence reaction, D-Luciferin is oxidized by luciferase to produce light. This is one of the most efficient light production systems. It can detect as few as 2000 luciferase molecules. It is also less susceptible to interference.

[0066] Coronavirus has two proteases targeting unique amino acid sequences with 3CL as the predominant one. Firefly luciferase can be modified by inserting a 3CL cleavage site within the luciferase sequence. The cleavage of the modified luciferase leads to inactivation of the enzyme. Thus, reduction in signal compared to control indicates presence of 3CL enzyme. Additionally, 3CL is present only in infected cells, not the virus. Thus, the assay detects active infection.

[0067] The firefly luciferase gene sequence containing the 3CL cleavage site can be expressed in E. coli as a recombinant protein. This recombinant firefly luciferase containing the 3CL cleavage site is named mutant luciferase or mLuc in this example. Construction, cloning and expression of recombinant proteins in E. coli is well known to those skilled in the art. mLuc was constructed, cloned into an appropriate vector and expressed in E. coli according to methods available in the literature, using commercially available reagents.

[0068] To test whether the recombinant mutant luciferase (mLuc) with 3CL cleavage site could be properly cleaved with 3CL protease, the recombinant mLuc was mixed with a recombinant COVID-19 viral 3CL enzyme and incubated at 37° C. Aliquots were removed at 0, 5, 10, 15, 20, 25 and 30 minutes after initiating the reaction, and were stopped immediately after removal by heat inactivation in a solution with SDS. The resulting reaction solution were resolved on an SDS-PAGE gel, followed by staining to visualize the proteins on the gel.

[0069] As shown in FIG. 6, significant cleavage was evident after 5 minutes. The mLuc is larger than the wild type Luc in molecular weight. Cleavage by 3CL enzyme resulted in two fragments, Fragments 1 and 2, as expected, demonstrating that mLuc can be cleaved by the 3CL enzyme.

Example 2: a Biochemiluminescent Assay for Detection of Cronovirus 3Cl Activity

[0070] In this example, mLuc described in Example 1 was used in an assay for detection of coronavirus 3CL activity. This assay consisted of two reagents, Reagent I and Reagent II. Reagent I contained ingredients that enable 3CL cleavage while Reagent II contained ingredients that enable firefly luciferase biochemiluminescent reaction.

[0071] A commercially available feline vaccine produced in culture cells was serially diluted and used in the experiments in this example. Since the vaccine contained ingredients from the cells, the samples also contained 3CL enzyme in the sample. The samples were first mixed with Reagent I and incubated at room temperature for 15 minutes along with a negative control, which contained no 3CL enzyme. After incubation, the reactions were mixed with Reagent II and immediately placed in a luminometer to measure the light signal. Relative light units were recorded.

[0072] For comparison, the diluted samples were also tested with real time RT-PCR.

[0073] The test results are shown in Table 1. As expected, the light signal increased as the samples were more diluted, which decreased the concentration of 3CL enzyme in the samples. On the other hand, the light signal of the samples relative to the negative control (the relative activity (RA)), was inversely related to the dilution factors. Based on extrapolation, a dilution of 1:106,739 would have given an RA of 1, which is the cut off value for detecting the coronavirus assay in this example. The extrapolated Ct value at 1:106,739 dilution is equivalent to detection sensitivity of 36 cycles of real time RT-PCR.

TABLE-US-00001 TABLE 1 Test Results of Serially Diluted Samples Containing the 3CL Enzyme Light Sample RT-PCR Signal Relative Dilution (average C.sub.t (Relative Activity Factor Value; n = 3) Light Unit) (RA) 1:10 18.22 46 19421 1:100 21.88 136 6569 1:1000 25.54 216 4136 1:10000 27.57 2612 342 Negative Control N/A 893,388 1

[0074] The method disclosed in this Example may be used to detect coronavirus infection. A clinical sample such as a throat swab or nasopharyngeal swab can be eluted in a sample buffer compatible with Reagent I. The ingredients in Reagent I may be prepared in, for example, 2× solution. The sample in sample buffer is mixed with 2× concentrated Reagent I in a 1:1 volume ratio. Incubate the reaction at room temperature for 15 minutes, followed by addition of equal volume of 3× Reagent II. The signal can be measured with a luminometer.

[0075] In some embodiments, the sample swabs are directly inserted into Reagent I and are left at room temperature for at least 15 minutes. The swabs are then removed from the Reagent I solution. After addition of Reagent II, the light signal is measured with a luminometer.

[0076] If the sample from a patient is positive with the assay described in the present invention, the sample proceeds immediately to confirm that positive test with a RT-PCR test to determine whether the sample contains COVID-19 virus. However, if the sample tests negative, there is no need for a RT-PCR test as the test has indicated that the patient is negative of all coronaviruses including COVID-19.

Example 3: A Biochemiluminescent Assay for Detection of Cronovirus 3Cl Activity in Purified COVID-19 Virus

[0077] The assay described in Example 2 was used to test the samples containing serially diluted purified COVID-19 virus at concentrations ranging from 0.1 to 10.sup.5 TCID.sub.50/mL. These virus samples were tested along with two positive controls, which contained recombinant 3CL enzyme, and one negative control. The light signal of the samples was compared to that of the negative control to derive the relative activity (RA). No 3CL activity was detected, indicating that coronavirus itself does not contain 3CL enzyme. The data is provided in FIG. 7.

Example 4: A Protease Enabled Signal Amplification (Pesa) Assay for Detection of Cronovirus 3Cl Activity

[0078] Aspects of this example are illustrated in FIG. 8.

[0079] In this example, firefly luciferase is used as the signal enabling molecule [1] and COVID-19 virus 3CL is used as the protease [6] for detection of COVID-19 virus infection. A recombinant fusion protein is constructed to contain the entire sequence of the firefly luciferase sequence in the N terminus, which is fused with the entire sequence of COVID-19 viral 3CL sequence [6] at the C terminus of the firefly luciferase [1]. Several amino acids in the virus sequence franking the N terminus of 3CL coding sequence is also introduced between the firefly luciferase and 3CL protein sequences as a linker sequence. This linker sequence [4] is derived from the COVID-19 virus pre-protein sequence. In some embodiment, this linker sequence is 4 amino acid sequence of AVLQ, which represents the amino acid sequence of alanine-valine-leucine-glutamine.

[0080] Another affinity moiety [10] can be added to the C terminus of 3CL sequence to further reduce background of 3CL enzyme in the recombinant fusion protein. Appropriate affinity moiety can be a His tag consisting of 6-8 amino acid histidine, which can bind to nickel ion. Another example of the affinity moiety is the streptavidin sequence, which can bind to biotin coated to a nanoparticle. A 3CL cleavage sequence needs to be introduced in front of affinity moiety [10] so that it can be cleaved by 3CL enzyme in the sample.

[0081] The nucleic acid sequence encoding the recombinant protein sequence can be cloned into an appropriate vector for expression in appropriate cells such as E. coli cells. After purification, the recombinant protein is used in a PESA assay for detection of 3CL activity in a sample. It is expected that there may be background activity due to low level of self-cleavage. A negative control is tested along with samples. The residual signal from the negative control is used the background signal, or commonly referred to as “noise”, to calculate the signal to noise ratio or S/N, which is the signal intensity in the sample divided by the signal intensity of the negative control. Presence of 3CL enzyme in the sample is indicated when the signal to noise or S/N exceeds 1.5, 2.0, 3.0, 4.0 or 5.0 or another threshold value.