Patent Description:
Currently, there are mainly two types of conventional virus pretreatment solutions: a Hank's matrix-based pretreatment solution and a guanidine salt-based pretreatment solution. The Hank's solution is a common balanced salt solution (BSS) for virus transport. Due to various factors, the Hank's solution can preserve a respiratory virus (such as an influenza virus, a novel coronavirus, etc.) for only a few hours. After a few hours, the morphology of the virus can be affected, which can affect the efficiency of detecting a viral nucleic acid. After an extended time, bacteria and fungi can easily grow, which lowers the pH value of the Hank's solution and accelerates degradation of the virus. Therefore, it is difficult to use the Hank's solution for long-term preservation of the virus and detection of a viral nucleic acid. The concentration of guanidine hydrochloride or Rnasin in the guanidine salt-based virus pretreatment solution is relatively high (<NUM> to <NUM>). A high concentration of a guanidine salt is suitable for virus inactivation at room temperature. However, the high concentration of the guanidine salt greatly inhibits nucleic acid extraction or purification (such as based on magnetic beads), the implementation of which requires multiple times of washing or the use of a spin column.

The extraction free nucleic acid release and amplification technology (EFNART), simply referred to as the "one-step" technique, refers to directly performing a nucleic acid amplification test for a sample by directly combining a sample nucleic acid releasing agent with a strong alkaline property and a highly compatible amplification system in a circumstance where no nucleic acid extraction or purification of the sample is required. Operations performed based on the "one-step method" will greatly reduce time for detecting a viral nucleic acid, particularly for an RNA virus. The present invention is expected to save time by <NUM>% or more and improve detection efficiency by <NUM>% in comparison with a traditional amplification method performed after nucleic acid extraction.

In the event of a major epidemic (for example, the <NUM> novel coronavirus (<NUM>-nCoV) epidemic that occurred from <NUM> to <NUM>), detection based on the one-step method is preferred because it saves detection time, improves efficiency, and will greatly contribute to controlling the epidemic.

However, existing common virus pretreatment solutions, such as the Hank's pretreatment solution and the guanidine salt-based pretreatment solution, cannot be well used for subsequent detection based on the one-step method. Thus, it is difficult to apply the existing common virus pretreatment solutions to scenarios in which rapid detection and screening of virus samples are required.

Therefore, there is a need in the art for a sample pretreatment solution that can be adequate for use in detection of a viral nucleic acid based on the one-step method. <CIT> is an example of a method and a kit for extracting adenovirus DNA from sputum samples. The buffer comprises <NUM> Tris-HCl at pH <NUM>, <NUM> EDTA at pH <NUM>, <NUM> NaCl, <NUM>% SDS, and <NUM>/ml proteinase K. The present invention's pretreatment solution has increased amplification efficiency compared to the buffer in <CIT>.

In view of this, in a first aspect, the present invention provides a pretreatment method of detecting a nucleic acid of a virus, the method comprising: mixing a sample stored in a pretreatment solution with a nucleic acid releasing agent and a qPCR amplification reagent, wherein the pretreatment solution comprises: Tris-HCl, EDTA-2Na, sodium chloride, a ribonuclease (RNase) inhibitor, and an antibiotic;.

The antibiotic comprises, but is not limited to, Proclin antibiotics (such as Proclin <NUM> and Proclin <NUM>) and NaN<NUM>.

For example, in a case where Proclin <NUM> is used as the antibiotic, the concentration of Proclin <NUM> may be about <NUM>% (v/v). For another example, in a case where Proclin <NUM> is used as the antibiotic, the concentration of Proclin <NUM> may be about <NUM>% (v/v), but the present invention is not limited thereto.

In a specific embodiment, the pretreatment solution comprises Tris-HCl at a concentration of <NUM>, EDTA-2Na at a concentration of <NUM>, sodium chloride at a concentration of <NUM>% (w/v), the RNase inhibitor at a concentration of <NUM> U/mL, and Proclin <NUM> at a concentration of <NUM>% (v/v).

The aforementioned pretreatment solution is adjusted to a pH value of <NUM>.

As used herein, the term "sample" refers to a sample that may contain a virus. The sample may be derived from human or animal blood, feces, urine, oral epithelial cells, exfoliated cells, buccal swabs, throat swabs, etc..

The term "pretreatment" mentioned in the present invention refers to treatment prior to performing a test for a sample, particularly treatment prior to detection of a nucleic acid of the sample (performed based on the one-step method).

The "pretreatment solution" mentioned in the present invention refers to a liquid for pretreating a virus sample.

In the present invention, the virus may be a DNA virus or an RNA virus.

In a preferred embodiment, the virus is an RNA virus.

In a more preferred embodiment, the virus is a coronavirus (such as the <NUM>-nCoV), a respiratory syncytial virus, and an enterovirus.

A nucleic acid detection reaction solution prepared by the pretreatment method of the present invention can be used to directly perform qPCR, without requiring an extraction or purification process, thereby improving detection efficiency and reducing detection time. The antibiotic and the RNase inhibitor employed in the present invention can be used for pretreatment of a DNA virus or an RNA virus, and can prevent the activity of a preserved virus from being affected by various microorganisms, such as bacteria, growing at room temperature. The RNA virus is more easily degraded due to the ubiquitous RNase. Damage to RNA that is caused by samples (various sample types such as oral epithelial cells, exfoliated cells, throat swabs, etc.) or sampling consumables in a medical sampling process is avoided while the method of the present invention is used, which is very conducive to long-term preservation and detection of the RNA virus.

It should be noted that in a case where the sample treated with the pretreatment solution of the present invention is not stored but directly (i.e., <NUM> hr after treatment) subjected to EFNART-based detection, as confirmed in the examples below, components of the pretreatment solution of the present invention have an effect of enhancing RT-PCT.

In a second aspect, the present invention provides a method of detecting a viral nucleic acid in a sample, comprising: directly performing detection by using the reaction solution prepared by the aforementioned method to perform qPCR amplification.

A sample releasing agent refers to a chemical agent that can release a nucleic acid in a sample, for example, a chemical agent with strong acidity or strong basicity. An exemplary sample releasing agent may be one or several of components comprising <NUM>-<NUM> mmol/L surfactin, <NUM>-<NUM> mmol/L potassium chloride, <NUM>-<NUM> mmol/L lithium chloride, triethanolamine dodecyl sulfate with a mass/volume ratio of <NUM>-<NUM>%, ethyl phenyl polyethylene glycol (NP-<NUM>) with a volume/volume ratio of <NUM>-<NUM>%, sodium dodecyl sulfonate with a mass/volume ratio of <NUM>-<NUM>%, ethanol with a volume/volume ratio of <NUM>-<NUM>%, etc., but the present invention is not limited thereto.

A qPCR amplification reagent refers to a reagent for a real-time quantitative nucleic acid amplification test. It can be understood by those skilled in the art that the qPCR amplification reagent usually contains DNA polymerase, dNTP, a PCR buffer solution, etc. For example, when RNA detection is to be performed, reverse transcriptase may also be further comprised. It is not difficult to understand that those skilled in the art can determine the components and concentration of a PCR reaction reagent according to specific needs (for example, the type and the content of a virus, etc.).

The term "one-step" mentioned in the present invention refers to the extraction free nucleic acid release and amplification technology (EFNART). The EFNART refers to directly performing a nucleic acid amplification test for a sample by directly combining a sample nucleic acid releasing agent with strong basicity and a highly compatible amplification system in a circumstance where no nucleic acid extraction or purification of the sample is required.

When detection of a viral nucleic acid in the sample is performed based on the one-step method, the sample in the preservation solution or pretreatment solution, the sample releasing agent, and the qPCR reaction solution are directly amplified after being mixed. Mixing may be performed at a general ratio in the art. In an exemplary embodiment, the sample in the preservation solution or the pretreatment solution, the sample releasing agent, and the qPCR reaction solution may be present in the ratio of about <NUM>:<NUM>:<NUM>, about <NUM>:<NUM>:<NUM>, or about <NUM>:<NUM>:<NUM> (v/v), but the present invention is not limited thereto.

In a third aspect, the present invention provides a pretreatment solution for detecting a nucleic acid of a virus, comprising: Tris-HCl, EDTA-2Na, sodium chloride, an RNase inhibitor, and an antibiotic,.

In some specific embodiments, the virus may be a DNA virus or an RNA virus.

In a fourth aspect, the present invention provides use of a pretreatment solution in preparation of a kit for detecting a virus by employing nucleic acid amplification based on the one-step method.

In a fifth aspect, the present invention provides a kit for detecting a viral nucleic acid based on the one-step method, the kit comprising the aforementioned pretreatment solution.

Further, the kit further comprises a sample releasing agent and a qPCR amplification reagent.

<FIG> is a graph of the results of "One Step" testing of the nucleic acid of the <NUM>-nCoV samples gradient-diluted by using the pretreatment method of the present invention and after being preserved at room temperature for <NUM> hr.

The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented therefrom. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, but not to limit the present invention.

In order to evaluate the virus pretreatment solution in the present invention, comparative analysis was performed on the virus pretreatment solution of the present invention (Tris-HCl at a concentration of <NUM>, EDTA-2Na at a concentration of <NUM>, sodium chloride at a concentration of <NUM>% (w/v), RNasin at a concentration of <NUM> U/mL, and Proclin <NUM> at a concentration of <NUM>% (v/v)), a saline solution, and commercially available virus pretreatment solutions. A comparison method was employed by performing dilution (<NUM>:<NUM>, v/v) pretreatment on an RSV throat swab sample that was clinically diagnosed to be positive. Direct amplification of the sample was performed at <NUM> hr, <NUM> hr, <NUM> hr, and <NUM> hr of the pretreatment time respectively under a pretreatment condition of a room temperature of <NUM>. The detection efficiency of real-time quantitative PCR (real-time qPCR) under the room temperature pretreatment condition was compared by Ct values to evaluate the effects of different pretreatment solutions on virus pretreatment. A qPCR amplification test was employed by using the EFNART "one-step" technique, such that a real-time qPCR amplification test was directly performed in a PCR amplification tube at a ratio of a pretreatment solution with the sample: a nucleic acid releasing agent: a PCR amplification reagent of <NUM>:<NUM>:<NUM>.

Real-time qPCR amplification testing procedures are as shown in Table <NUM>, and the results are as shown in Table <NUM>.

The aforementioned results showed that pretreatment was performed with the virus pretreatment method in the present invention at room temperature for <NUM> hr, and the pretreatment method can adequately pretreat the gradient-diluted RSV sample. In addition, testing could be directly performed by means of EFNART. After pretreatment was performed for a long time based on a saline solution matrix, the Ct values could be delayed, and the nucleic acid of the virus was degraded to a certain extent, which affected the amplification efficiency. In contrast, the Hank's pretreatment solution and the guanidine salt pretreatment solution, which are commonly used and commercially available, cannot provide an effective way of performing virus pretreatment and a PCR amplification test in this scheme.

In order to evaluate the virus pretreatment solution in the present invention, comparative analysis was performed on the virus pretreatment solution of the present invention (Tris-HCl at a concentration of <NUM>, EDTA-2Na at a concentration of <NUM>, sodium chloride at a concentration of <NUM>% (w/v), RNasin at a concentration of <NUM> U/mL, and Proclin <NUM> at a concentration of <NUM>% (v/v)), a saline solution, and commercially available virus pretreatment solutions. A comparison method was employed by performing dilution (<NUM>:<NUM>, v/v) pretreatment on a <NUM>-nCoV nucleic acid that was clinically diagnosed to be positive. Direct amplification of the sample was performed at <NUM> hr, <NUM> hr, <NUM> hr, and <NUM> hr of the pretreatment time respectively under the pretreatment condition of a room temperature of <NUM>. The testing efficiency of real-time qPCR under the room temperature pretreatment condition was compared by Ct values to evaluate the effects of different pretreatment solutions on virus pretreatment. A qPCR amplification test was employed by using the EFNART "one-step" technique, such that a real-time qPCR amplification test was directly performed in a PCR amplification tube at a ratio of a pretreatment solution with the nucleic acid: a nucleic acid releasing agent: a PCR amplification reagent of <NUM>: <NUM>:<NUM>. The results are as shown in Table <NUM>. The <NUM>-nCoV nucleic acid gradient-diluted by using the virus pretreatment method in the present invention was stored at room temperature for <NUM> hr. The results of the "One Step" testing are as shown in <FIG>.

The aforementioned results showed that pretreatment was performed with the virus pretreatment method in the present invention at room temperature for <NUM> hr, and the pretreatment method can adequately pretreat the gradient-diluted <NUM>-nCoV nucleic acid. In addition, testing could be directly performed by means of EFNART. After pretreatment was performed for a long time based on a saline solution matrix, the Ct values could be delayed, and the nucleic acid of the virus was degraded to a certain extent, which affected the amplification efficiency. In contrast, the Hank's pretreatment solution and the guanidine salt pretreatment solution, which are commonly used and commercially available, cannot provide an effective way of performing virus pretreatment and a PCR amplification test in this scheme.

In order to evaluate the virus pretreatment solution in the present invention, comparative analysis was performed on the virus pretreatment solution of the present invention (Tris-HCl at a concentraion of <NUM>, EDTA-2Na at a concentraion of <NUM>, sodium chloride at a concentraion of <NUM>% (w/v), SDS at a concentraion of <NUM>%, and Proclin <NUM> at a concentraion of <NUM>% (v/v)), a saline solution, and commercially available virus pretreatment solutions. A comparison method was employed by performing dilution (<NUM>:<NUM>, v/v) pretreatment on an EV general type throat swab sample that was clinically diagnosed to be positive. Direct amplification of the sample was performed at <NUM> hr, <NUM> hr, <NUM> hr, and <NUM> hr of the pretreatment time respectively under the pretreatment condition of a room temperature of <NUM>. The detection efficiency of real-time qPCR under the room temperature pretreatment condition was compared by Ct values to evaluate the effects of different pretreatment solutions on virus pretreatment. A qPCR amplification test was employed by using the EFNART "one-step" technique, such that a real-time qPCR amplification test was directly performed in a PCR amplification tube at a ratio of a pretreatment solution with the sample: a nucleic acid releasing agent: a PCR amplification reagent of <NUM>:<NUM>:<NUM>. The results are as shown in Table <NUM>:.

The aforementioned results showed that pretreatment was performed with the virus pretreatment method in the present invention at room temperature for <NUM> hr, and the pretreatment method can adequately pretreat the gradient-diluted EV sample. In addition, testing could be directly performed by means of EFNART. After pretreatment was performed for a long time based on a saline solution matrix, the Ct values could be delayed, and the nucleic acid of the virus was degraded to a certain extent, which affected the amplification efficiency. In contrast, the Hank's pretreatment solution and the guanidine salt pretreatment solution, which are commonly used and commercially available, cannot provide an effective way of performing virus pretreatment and a PCR amplification test in this scheme.

Compared with DNA viruses, pretreatment and detection of RNA viruses are more susceptible to environmental factors due to higher requirements. A pretreatment process involving the RNase contained in consumables has a particularly vital impact on testing of the RNA virus. In order to evaluate the pretreatment effect of the virus pretreatment solution in the present invention (tTris-HCl at a concentraion of <NUM>, EDTA-2Na at a concentraion of <NUM>, sodium chloride at a concentraion of <NUM>% (w/v), Rnasin at a concentraion of <NUM> U/mL, and Proclin <NUM> at a concentraion of <NUM>% (v/v)) on the RNA virus, the nucleic acid extracted from a <NUM>-nCoV sample pretreated with the virus pretreatment solution of the present invention was divided into two parts (A/B). At the same time, a nucleic acid (C) extracted from a <NUM>-nCoV sample with a same concentration that was pretreated in the saline solution was prepared. In addition, <NUM>µg/mL RNase A was respectively added into solution A and solution C. The three solutions containing the <NUM>-nCoV nucleic acid were pretreated at room temperature (<NUM>) for <NUM> hr. Then the RNA virus was pretreated and tested with the pretreatment solution in the present invention by adopting the EFNART "one-step" technique. Detection was conducted by directly performing a real-time qPCR amplification test in a PCR amplification tube at a ratio of a pretreatment solution with the nucleic acid: a nucleic acid releasing agent: a PCR amplification reagent of <NUM>:<NUM>:<NUM>. The results are as shown in Table <NUM>.

It was proved by the experiments that in a case where pretreated nucleic acid sample D was taken as the reference, addition of <NUM>µg/mL RNase A in the present invention did not affect the effect of detecting a nucleic acid of <NUM>-nCoV, and effective components in the present invention could digest the RNase, thus reducing the effect of the RNase on the experimental testing, so that the efficiency of direct detection of the RNA virus could be guaranteed. Judging from pretreated nucleic acid sample C, addition of the <NUM>µg/mL RNase A included in the experimental conditions could digest and degrade the RNA in an experiment, thereby greatly affecting direct detection of the RNA virus and leading to risk of misses.

In order to evaluate the effect of the virus pretreatment solution in the present invention on DNA virus preservation and amplification testing, comparative analysis was performed on the virus pretreatment solution of the present invention (Tris-HCl at a concentration of <NUM>, EDTA-2Na at a concentration of <NUM>, sodium chloride at a concentration of <NUM>% (w/v), RNasin at a concentration of <NUM> U/mL, and Proclin <NUM> at a concentration of <NUM>% (v/v)), a saline solution, and commercially available virus pretreatment solutions. A comparison method was employed by performing dilution (<NUM>:<NUM>, v/v) pretreatment on an HBV serum sample that was clinically diagnosed to be positive. Direct amplification of the sample was performed at <NUM> hr, <NUM> hr, <NUM> hr, and <NUM> hr of the pretreatment time respectively under the pretreatment condition of a room temperature of <NUM>. The testing efficiency of real-time qPCR under the room temperature pretreatment condition was compared by Ct values to evaluate the effects of different pretreatment solutions on virus pretreatment. A qPCR amplification test was employed by using the EFNART "one-step" technique, such that a real-time qPCR amplification test was directly performed in a PCR amplification tube at a ratio of a pretreatment solution with the DNA virus: a nucleic acid releasing agent: a PCR amplification reagent of <NUM>:<NUM>:<NUM>. The results are as shown in Table <NUM>.

The aforementioned results showed that although the original design of the virus pretreatment method in the present invention was aimed at virus matrix preservation for RNA viruses for performing amplification based on the "one-step method", the virus pretreatment method can also be used for preservation and detection of DNA viruses by performing amplification based on the one-step method.

In order to verify the effectiveness of various components in the present invention, after relevant components in the present invention were adjusted and reduced, the sample was preserved and was subjected to pretreatment and a comparison experiment. Separately optimized components included an RNase inhibitor, an antibiotic, EDTA-2Na, etc. Concentrations of the adjusted various components are as shown in Table <NUM>. qPCR amplification testing was directly performed after RSV was preserved with the pretreatment solutions at room temperature for <NUM> hr that were prepared at different concentrations, and the detection efficiency of real-time qPCR under the room temperature pretreatment condition was compared by Ct values to evaluate the effects of different pretreatment solutions on virus pretreatment. A qPCR amplification test was employed by using the EFNART "one-step" technique, such that a real-time qPCR amplification test was directly performed in a PCR amplification tube at a ratio of a pretreatment solution with the sample: a nucleic acid releasing agent: a PCR amplification reagent of <NUM>: <NUM>:<NUM>.

Claim 1:
A pretreatment method of detecting a nucleic acid of a virus, the method comprising:
mixing a sample stored in a pretreatment solution with a nucleic acid releasing agent and a qPCR amplification reagent, wherein the pretreatment solution comprises Tris-HCl, EDTA-2Na, sodium chloride, a ribonuclease (RNase) inhibitor, and an antibiotic;
wherein the concentration of Tris-HCl is <NUM> to <NUM>, the concentration of EDTA-2Na is <NUM> to <NUM>, the concentration of sodium chloride is <NUM>% (w/v) to <NUM>% (w/v), the concentration of the RNase inhibitor is <NUM> U/mL to <NUM> U/mL, and the concentration of the antibiotic is <NUM>% to <NUM>% in the pretreatment solution; and
wherein the pretreatment solution has a pH of <NUM>-<NUM>.