CN112111599A

CN112111599A - Multiple detection kit and method for drogong virus and zika virus

Info

Publication number: CN112111599A
Application number: CN202010559238.3A
Authority: CN
Inventors: 林猷斌; 张微石; 李家俊
Original assignee: Delta Electronics International Singapore Pte Ltd
Current assignee: Delta Electronics International Singapore Pte Ltd
Priority date: 2019-06-19
Filing date: 2020-06-18
Publication date: 2020-12-22
Also published as: TW202115262A; TWI754967B

Abstract

The multiple detection kit for the drogonella virus and the zika virus comprises a first primer group with specificity to the drogonella virus and a second primer group with specificity to the zika virus. The first primer set includes a forward primer having a sequence of SEQ ID NO. 1 and a reverse primer having a sequence of SEQ ID NO. 2. The second primer set includes a forward primer having a sequence of SEQ ID NO. 4, a forward primer having a sequence of SEQ ID NO. 5, and a reverse primer having a sequence of SEQ ID NO. 6. The kit also comprises a probe which is specific to the Tohrysovirus and has a sequence of SEQ ID NO. 3, and a probe which is specific to the Zirca virus and has a sequence of SEQ ID NO. 7. The kit further comprises an internal control template of RNA obtained by in vitro transcription of the proA gene of the soft rot bacteria, a forward primer with a sequence of SEQ ID NO. 8, a reverse primer with a sequence of SEQ ID NO. 9 and a probe with a sequence of SEQ ID NO. 10.

Description

Multiple detection kit and method for drogong virus and zika virus

[ technical field ] A method for producing a semiconductor device

The present application relates to multiplex assays for drogonella virus and zika virus, and more particularly to a kit and method for multiplex assays for drogonella virus and zika virus.

[ Prior Art ] A method for producing a semiconductor device

Drogonella virus (Chikungunya virus, CHIKV) is an arbovirus and belongs taxonomically to the genus Alphavirus (Alphavirus) and the family of Togaviridae. This virus is transmitted from person to person via the bite of virus-carrying spotted mosquitoes (Aedes mosquitoes). This virus was first isolated from serum and spotted mosquitoes in 1953.

Flexor disease usually occurs in africa and asia, but epidemic situations have also developed in europe and america due to climate change and globalization. The symptoms of the disease range from asymptomatic to extremely pathological. Drogue disease usually begins with an acute febrile phase up to 40 ℃ and lasts for several days to one week, followed by long-term joint disease, affecting the joints of the extremities, and pain caused by drogue virus infection of the joints lasts for weeks or months. It is believed that the term "flexor" derives from the local dialect of africa (makangde "bend"), describing the distorted posture of a patient as a result of suffering from extreme joint pain. Other non-specific symptoms may include headache, conjunctivitis, digestive discomfort, and slight photophobia.

Zika virus (Zika virus) is a mosquito-transmitted flavivirus that was first discovered in monkeys in 1947, and later in humans in 1952 in the united kingdom of udda and tanzania. This virus is transmitted from person to person via the bite of virus-carrying spotted mosquitoes (Aedes mosquitoes).

Before 2015, outbreaks of zika virus typically occurred in africa, southeast asia and the pacific islands. However, due to globalization, the most severe epidemic occurred 3 months in 2015, which began to outbreak from brazil and soon spread to america, africa, asia and europe. The symptoms of the disease range from asymptomatic to mild symptoms, such as fever, rash, headache, joint pain, and conjunctivitis and muscle pain. Often, mild symptoms last from a few days to a week, and an infected person may not even realize that himself has been infected. However, according to recent findings, zirca virus infection can cause neurological diseases, such as Guillain-barre syndrome (Guillain-barre syndrome) and a severe congenital defect known as congenital Zika syndrome (congenital Zika syndrome), which has the most obvious symptom of severe microcephaly in newborns of infected mothers.

Three techniques currently used for diagnosis of these viruses are virus isolation (against zirca virus), serological detection, and real-time reverse transcription polymerase chain reaction (real-time RT-PCR), which confirm viral infection by virus detection, antibody detection specific to virus, and virus RNA detection, respectively. Virus isolation was performed by using live virus in cell culture, observing cytopathic effect (CPE) caused by the virus, and neutralizing CPE with antiserum specific to the virus of drogonella virus to confirm CPE. The main drawback of this diagnostic method is that it must be performed in a class 3 biosafety laboratory and takes 1-2 weeks to complete the test. Serological tests, while providing a definitive result within 15 minutes to 2-3 days, involve the detection of IgM and IgG antibodies in the patient's serum, which appear only after several days of clinical symptoms (e.g., fever). In addition, the specificity of serological tests is still questioned due to the possibility of false positives due to cross-reactivity of other arboviruses with IgM, especially if the patient had a previous history of infection with other flaviviruses. Real-time reverse transcription polymerase chain reaction is a relatively new diagnostic method that can solve the disadvantages of the above methods. Due to the higher sensitivity, real-time reverse transcription polymerase chain reaction can detect the genome of the drogonella virus and the genome of the zika virus more quickly even in the early stage of infection, and the probability of false positive is lower due to the higher specificity.

Although the sensitivity and specificity of the detection of the Tourette and Zirca viruses has improved in recent years, the speed provided by current real-time reverse transcription polymerase chain reaction detection is still insufficient because the typical reverse transcription polymerase chain reaction processing time is almost one-half hour. Therefore, there is still a strong need to provide a Point-of-Care (POC) diagnostic method for detecting the virus of the drogonella virus and the zirca virus sensitively and specifically and providing a faster processing time.

[ summary of the invention ]

An object of the present invention is to provide a multiplex detection of drogonella virus and zika virus, which has high sensitivity, high specificity and a shortened reaction time.

It is another object of the present invention to provide a multiplex detection of drogonvirus and zika virus for simultaneous detection of drogonvirus and zika virus in the same reaction.

To achieve the above objective, one embodiment of the present disclosure provides a multiple detection kit for a virus of drogonella and zika virus, comprising at least one of a first primer set specific to the virus of drogonella and a second primer set specific to the virus of zika. The first primer set is selected from: (a) a forward primer having the nucleotide sequence of SEQ ID NO. 1 and a reverse primer having the nucleotide sequence of SEQ ID NO. 2; and (b) a forward primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 1 and a reverse primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 2. The second primer set is selected from: (a) a forward primer having the nucleotide sequence of SEQ ID NO. 4 and a reverse primer having the nucleotide sequence of SEQ ID NO. 6; (b) a forward primer having the nucleotide sequence of SEQ ID NO. 5 and a reverse primer having the nucleotide sequence of SEQ ID NO. 6; (c) a first forward primer having the nucleotide sequence of SEQ ID NO. 4, a second forward primer having the nucleotide sequence of SEQ ID NO. 5, and a reverse primer having the nucleotide sequence of SEQ ID NO. 6; and (d) at least one forward primer having a nucleotide sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO. 4 and SEQ ID NO. 5 and a reverse primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 6.

In one embodiment, the multiplex assay kit further comprises a probe specific for the droguet virus, wherein the probe has a nucleotide sequence selected from the group consisting of: (a) 3, the nucleotide sequence of SEQ ID NO; (b) a nucleotide sequence complementary to SEQ ID NO. 3; and (c) a nucleotide sequence having at least 90% identity to SEQ ID NO. 3 or the complement thereof. The 5 'end of the probe is provided with a reporter dye, and the 3' end is provided with a quencher.

In one embodiment, the multiplex assay kit further comprises a probe specific for zirca virus, wherein the probe has a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO. 7; (b) a nucleotide sequence complementary to SEQ ID NO. 7; and (c) a nucleotide sequence having at least 90% identity to SEQ ID NO 7 or the complement thereof. The 5 'end of the probe is provided with a reporter dye, and the 3' end is provided with a quencher.

In one embodiment, the multiplex assay kit further comprises an internal control template, which is RNA obtained by in vitro transcription of the proA gene of a soft rot bacterium (Pebacter carotovorum).

In one embodiment, the multiplex assay kit further comprises a control primer set specific for the proA gene, wherein the control primer set is selected from the group consisting of: (a) a forward primer having the nucleotide sequence of SEQ ID NO. 8 and a reverse primer having the nucleotide sequence of SEQ ID NO. 9; and (b) a forward primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 8 and a reverse primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 9.

In one embodiment, the multiplex assay kit further comprises a probe specific for the proA gene, wherein the probe has a nucleotide sequence selected from the group consisting of: (a) 10, the nucleotide sequence of SEQ ID NO; (b) a nucleotide sequence complementary to SEQ ID NO. 10; and (c) a nucleotide sequence having at least 90% identity to SEQ ID NO 10 or the complement thereof. The 5 'end of the probe is provided with a reporter dye, and the 3' end is provided with a quencher.

To achieve the above objects, another embodiment of the present invention provides a method for multiplex detection of a virus of drogonella and zika, comprising the steps of: (a) contacting a biological sample with the multiplex assay kit; (b) amplifying nucleic acid of the virus of drogonella virus and/or zika virus from the biological sample; and (c) detecting the presence of amplified nucleic acid of the virus.

In one embodiment, the method further comprises a step of extracting nucleic acid from the biological sample before step (a).

In one embodiment, step (b) of the method includes the sub-steps of: (b1) reverse transcribing the viral genomic RNA into complementary DNA; and (b2) amplifying the complementary DNA using a real-time polymerase chain reaction.

The terms "a" and "an" when used in conjunction with the terms "including" and/or "comprising" in the claims and/or the specification may mean "one," but are also consistent with the meaning of "one or more," at least one, "and" one or more than one.

The term "or" in the claims means "and/or" unless explicitly indicated to refer only to alternatives or alternatives are mutually exclusive, even though the disclosure supports definitions referring only to alternatives and "and/or".

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[ description of the drawings ]

FIG. 1 shows the partial cDNA sequence of the non-structural protein 1 gene of the drogonella virus, and the binding positions of the forward primer, the reverse primer and the probe on the sequence.

FIG. 2 shows a partial cDNA sequence of an envelope gene of a Z-card virus, and binding sites of a forward primer, a reverse primer and a probe on the sequence.

FIG. 3 shows the partial cDNA sequence of the proA gene of soft rot bacteria, and the binding positions of the forward primer, the reverse primer and the probe on the sequence.

FIGS. 4 and 5 show the results of the analysis of the specificity of the Tourette virus and the Zirca virus, respectively.

FIGS. 6A and 6B show the results of joint detection of the Tourette virus and the Zika virus.

FIG. 7 shows amplification curves generated from different copy numbers of drogue genomic RNA.

FIG. 8 shows the results of FIG. 7 resulting in a drogue test standard curve.

FIG. 9 shows amplification curves generated from different copy numbers of the genomic RNA of Zka.

FIG. 10 shows a calibration curve for the detection of the Zehnder device resulting from the results of FIG. 9.

FIG. 11 shows a comparison of the multiplex assays of the present case with two commercially available drouth assay kits.

FIG. 12 shows a comparison of the multiplex assay of the present case with two commercial Zrcard assay kits.

[ embodiment ] A method for producing a semiconductor device

Some embodiments that embody the features and advantages of this disclosure will be described in detail in the description that follows. It will be understood that the present disclosure is capable of various modifications without departing from the scope of the disclosure, and that the description and drawings are to be taken in an illustrative rather than a limiting sense.

The present disclosure provides multiplex detection of drogonvirus and zika virus for simultaneous detection of drogonvirus and zika virus with high sensitivity, high specificity, and reduced reaction time. In the embodiment of the present invention, real-time reverse transcription polymerase chain reaction (real-time RT-PCR), also called quantitative reverse transcription polymerase chain reaction (qRT-PCR), is used in combination with a probe-based detection system to rapidly, sensitively and specifically diagnose human viral infection. The real-time reverse transcription polymerase chain reaction (real-time RT-PCR) mainly includes two steps, the first step is to reverse-transcribe genomic RNA (genomic RNA) of the virus of. In particular, in real-time polymerase chain reaction (real-time PCR), specific forward, reverse and probe primers hybridize to the cDNA target of the virus, i.e., the virus or zirca virus, wherein the probe is terminated with a reporter dye (reporter dye) at the 5 'end and a quencher (quencher) at the 3' end. During the PCR amplification reaction, the probe is cleaved, allowing the reporter dye to separate from the quencher, and the fluorescence emitted by the reporter dye is detected.

The embodiment of the scheme provides a drogong virus detection kit and a zika virus detection kit, wherein the two kits can be used independently to detect the drogong virus and the zika virus respectively, and can also be used together to be a multiple detection kit capable of detecting the drogong virus and the zika virus simultaneously. The design of the primers and probes used in these kits will be described in detail below.

The drogong virus is a single-stranded RNA virus with 11826 bases (S27 strain; NCBI reference sequence database NC-004162.2). In the present example, the nonstructural protein 1(nsP1) gene of the drogonella virus was selected as the detection target. The drogong virus detection kit comprises a forward primer and a reverse primer, and can additionally comprise a probe.

FIG. 1 shows the partial cDNA sequence (designated by SEQ ID NO:11, corresponding to positions 1051 to 1225 of the NC-004162.2 gene sequence) of the non-structural protein 1 gene of the drometrivirus, and the binding positions of the forward primer, the reverse primer and the probe on the non-structural protein 1 gene sequence. The forward primer has the nucleotide sequence of SEQ ID NO:1 (5'-GCGACCATTTGTGATCAAATGACC-3'), starting at position 1082 of the gene sequence, and is a 24-mer size. The reverse primer has the nucleotide sequence of SEQ ID NO. 2 (5'-GTTCTGCCGTTAACCACTATTCTCTG-3'), starting at position 1191 of the gene sequence, and has a size of 26-mer. The probe is a reverse probe having the nucleotide sequence of SEQ ID NO 3 (5'-AGCTTCTGTGCATCCTCCGGCGTGAC-3') starting at position 1149 of the gene sequence and being 26-mer in size. Using the forward and reverse primers described above will amplify an amplicon (amplicon) of size 110-bp.

The Zerca virus is a single-stranded RNA virus having 10675 bases (PRVABC59 virus strain; NCBI reference sequence database KU 501215.1). In the present embodiment, the envelope gene (E) of the Z-card virus is selected as the target of detection. The kit for detecting the zirca virus comprises at least one forward primer and one reverse primer, and can additionally comprise a probe.

FIG. 2 shows the partial cDNA sequence (denoted by SEQ ID NO:12, corresponding to positions 2101 to 2275 of the sequence of KU501215.1 gene) of the envelope gene of the Z-ka virus, and the binding positions of the forward primer, the reverse primer and the probe on the sequence of the envelope gene. The first forward primer has the nucleotide sequence of SEQ ID NO. 4 (5'-GGAGTGGCAGCACCATTG-3'), starting at position 2181 of the gene sequence and is 18-mer in size. The second forward primer has the nucleotide sequence of SEQ ID NO:5 (5'-GGAGTGGCAGTACCATTG-3'), also starting at position 2181 of the gene sequence and is 18-mer in size. The reverse primer has the nucleotide sequence of SEQ ID NO 6 (5'-CAGGCTGTGTCTCCCAAGA-3'), starting at position 2262 of the gene sequence and is 19-mer in size. The probe was a reverse probe having the nucleotide sequence of SEQ ID NO:7 (5'-TTCTCTTGGCACCTCTCACAGTGGCTTCAA-3') starting at position 2237 of the gene sequence and being a 30-mer size. Using the forward primer (having the sequence of SEQ ID NO:4 or SEQ ID NO:5) and the reverse primer described above will amplify an amplicon having a size of 82-bp.

The second forward primer having the sequence of SEQ ID NO. 5 has only one nucleotide change compared to the first forward primer having the sequence of SEQ ID NO. 4, and the first and second forward primers may be useful for detecting different Zka virus strains. In one embodiment, the Zirca virus detection kit comprises a first forward primer having the sequence of SEQ ID NO. 4 and a reverse primer having the sequence of SEQ ID NO. 6. In another embodiment, the Zirca virus detection kit comprises a second forward primer having the sequence of SEQ ID NO. 5, and a reverse primer having the sequence of SEQ ID NO. 6. Or in another embodiment, the Zirca virus detection kit comprises a first forward primer having the sequence of SEQ ID NO. 4, a second forward primer having the sequence of SEQ ID NO. 5, and a reverse primer having the sequence of SEQ ID NO. 6.

In order to confirm the specificity of the primers and probes used for the virus, each of the primers and probes (SEQ ID NOS: 1 to 3) was aligned to two databases of human genome transcription (human genomic plus transcript) and nucleotide collection (nucleotide collection) using NCBI BLAST system, and the alignment showed that NO other similar species had the same sequence fragment as the primers and probes designed in this case. The result shows that the primer and the probe designed by the scheme have very high specificity to the drogong virus and can be used for amplifying and detecting the non-structural protein 1 gene of the drogong virus. In one embodiment, the primers and probes can be used to amplify and detect only the non-structural protein 1 gene of drogonella virus.

To confirm the specificity of the primers and probes used for the zirca virus, each of the primers and probes (SEQ ID nos. 4 to 7) were aligned using the NCBI BLAST system against two databases of human genome transcription (human genomic plus transcript) and nucleotide collection (nucleotide collection), and the alignment revealed that NO other closely related species had identical sequence fragments to those of SEQ ID nos. 4, 5, and 7 designed in this case, whereas SEQ ID No. 6 had 100% similarity to the third serotype of Dengue virus (Dengue serotype 3). Since the primer and probe lines were tested in groups, the forward primer (SEQ ID NO:4 or 5) and the reverse probe (SEQ ID NO:7) did not bind to the genomic material of the third serotype of dengue virus, even though the reverse primer (SEQ ID NO:6) could bind to the genomic material of the third serotype of dengue virus. That is, the primer and probe combination of the present embodiment does not produce a false positive detection result for the third serotype of dengue virus, and can still specifically detect the envelope gene of the zka virus. In other words, the primers and probes designed by the scheme have very high specificity to the zirca virus, and can be used for amplifying and detecting envelope genes of the zirca virus. In one embodiment of the present disclosure, the primers and probes can be used only for amplifying and detecting the envelope gene of the zirca virus.

In addition, the present embodiment further provides a kit for detecting an internal control template (internal control template) which is RNA (in vitro transcribed RNA) obtained from the proA gene of a soft rot bacterium (Lactobacillus SCRI121 strain; NCBI reference sequence database HM 157163.1). The proA internal control detection kit comprises a forward primer and a reverse primer, and may additionally comprise a probe.

FIG. 3 shows the partial cDNA sequence of the proA gene (denoted by SEQ ID NO:13, corresponding to positions 291 to 490 of the HM157163.1 gene sequence) and the binding positions of the forward primer, the reverse primer and the probe on the proA gene sequence. The forward primer has the nucleotide sequence of SEQ ID NO:8 (5'-CTGCTGGTCAATCAACGTATCG-3'), starting at position 301 of the gene sequence and is a 22-mer in size. The reverse primer has the nucleotide sequence of SEQ ID NO:9 (5'-GGTCGTTAGAAAGCCATTCATCG-3') starting at position 478 of the gene sequence and is 23-mer in size. The probe was a forward probe having the nucleotide sequence of SEQ ID NO:10 (5'-TCCATGCCAGTCCTTCAGCGATGCCTTA-3'), starting at position 377 of the gene sequence and being 28-mer in size. The forward primer and the reverse primer can be used for amplifying to generate an amplicon with the size of 178-bp.

In one embodiment, the multiple testing kit for the drogonella virus and the zika virus comprises a drogonella virus testing kit, a zika virus testing kit and a proA internal control testing kit. The primers and probes used in this multiplex assay kit are shown in Table 1.

TABLE 1

In some embodiments, the primers and probes of the present disclosure are not limited to those having the exact same sequences as SEQ ID NO:1 to 10. It is noted that the sequence to be hybridized does not require perfect complementarity to provide a stable hybrid, and in many cases, a stable hybrid is formed when the base mismatch ratio is less than 10%. In addition, genetic variations may also exist for a particular droguet virus strain or zirca virus strain. Thus, primers and probes having sequences at least 90% identical to SEQ ID NO 1 to 10 may have similar specificity to the original sequence and thus may also be used for multiplex detection of the Cherovirus and the Zirca virus.

In some embodiments, since the primer pair consisting of the corresponding forward and reverse primers can specifically detect the Chevron virus (SEQ ID NOS: 1 and 2), the Zirca virus (SEQ ID NOS: 4 to 6), or the proA internal control (SEQ ID NOS: 8 and 9), the probe sequence can be designed as a probe sequence as long as it is located between the positions of the corresponding forward and reverse primers, and thus, the probe sequence is not limited to the aforementioned probe sequences (SEQ ID NOS: 3, 7 and 10). In addition, since the probe can be selectively hybridized to any strand of DNA, the complementary sequences at the same position can be used as probe sequences, and therefore, the sequences complementary to the aforementioned probe sequences (SEQ ID NOS: 3, 7 and 10) can also be used as probe sequences for detecting the internal controls of the Torpovirus, the Zucavirus and the proA, respectively.

According to the above, the present disclosure provides a multiple detection kit for a virus of the drogue virus and a virus of the zika virus, including at least one of a first primer set specific to the drogue virus and a second primer set specific to the virus of the zika virus. The first primer set is selected from: (a) a forward primer having the nucleotide sequence of SEQ ID NO. 1 and a reverse primer having the nucleotide sequence of SEQ ID NO. 2; and (b) a forward primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 1 and a reverse primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 2. The second primer set is selected from: (a) a forward primer having the nucleotide sequence of SEQ ID NO. 4 and a reverse primer having the nucleotide sequence of SEQ ID NO. 6; (b) a forward primer having the nucleotide sequence of SEQ ID NO. 5 and a reverse primer having the nucleotide sequence of SEQ ID NO. 6; (c) a first forward primer having the nucleotide sequence of SEQ ID NO. 4, a second forward primer having the nucleotide sequence of SEQ ID NO. 5, and a reverse primer having the nucleotide sequence of SEQ ID NO. 6; and (d) at least one forward primer having a nucleotide sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO. 4 and SEQ ID NO. 5 and a reverse primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 6.

In one embodiment, the multiplex assay kit for drogonella virus and zika virus further comprises a probe specific for drogonella virus, wherein the probe has a nucleotide sequence selected from the group consisting of: (a) 3, the nucleotide sequence of SEQ ID NO; (b) a nucleotide sequence complementary to SEQ ID NO. 3; and (c) a nucleotide sequence having at least 90% identity to SEQ ID NO. 3 or the complement thereof.

In one embodiment, the multiple test kit for the virus of the drogonella virus and the zika virus further comprises a probe specific for the zika virus, wherein the probe has a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO. 7; (b) a nucleotide sequence complementary to SEQ ID NO. 7; and (c) a nucleotide sequence having at least 90% identity to SEQ ID NO 7 or the complement thereof.

In one embodiment, the multiple test kit for the virus of the dromerin and zika further comprises an internal control template, which is an RNA obtained by in vitro transcription of the proA gene of a soft rot bacterium (Pebacter carotovorum). Accordingly, the multiple detection kit for the drogonvirus and the zika virus further comprises a control primer group specific to the proA gene, wherein the control primer group is selected from the group consisting of: (a) a forward primer having the nucleotide sequence of SEQ ID NO. 8 and a reverse primer having the nucleotide sequence of SEQ ID NO. 9; and (b) a forward primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 8 and a reverse primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 9. Furthermore, the multiple detection kit for the drogonvirus and zika virus further comprises a probe specific for the proA gene, wherein the probe has a nucleotide sequence selected from the group consisting of: (a) 10, the nucleotide sequence of SEQ ID NO; (b) a nucleotide sequence complementary to SEQ ID NO. 10; and (c) a nucleotide sequence having at least 90% identity to SEQ ID NO 10 or the complement thereof.

The specific primers and probes are designed to hybridize and amplify the target gene. Therefore, the present invention also provides a method for multiplex detection of a virus of the drogonella virus and a virus of the zika virus, the method comprising the steps of: (a) contacting a biological sample with the multiplex assay kit; (b) amplifying nucleic acid of the virus of drogonella virus and/or zika virus from the biological sample; and (c) detecting the presence of amplified nucleic acid of the virus.

In one embodiment, step (b) of the method includes the sub-steps of: (b1) reverse transcribing viral genomic RNA to complementary DNA; and (b2) amplifying the complementary DNA using a real-time polymerase chain reaction.

The probe for real-time polymerase chain reaction is a reporter dye attached to the 5 'terminus and a quencher attached to the 3' terminus. During the PCR amplification reaction, the probe is cleaved, allowing the reporter dye to separate from the quencher, and the fluorescence emitted by the reporter dye is detected. In the multiplex detection of different target genes, different probes for detecting the drogonvirus, zika virus and proA internal controls can be labeled with different reporter dyes having distinguishable fluorescent colors, which facilitates observation of the detection results. In one embodiment, the reporter dyes can be selected from, but not limited to, fluorescent dyes such as ROX, FAM, HEX, Cy5, TET, and Texas Red, respectively. In one embodiment, the quencher is a dark quencher (dark quencher), such as, but not limited to, an Iowa Black FQ quencher or a Black Hole quencher.

The primers and probes provided in the embodiments are optimized for rapid detection of the retrovirus and the zavirus through a real-time reverse transcription polymerase chain reaction (RT-PCR) performed in a fast cycling mode. Table 2 shows the temperature profile of real-time RT-PCR in the rapid cycling mode. In one embodiment, the fast cycling mode refers to the temperature profile of the real-time reverse transcription polymerase chain reaction as follows: (1) a reverse transcription hold period of 5 minutes at 42 ℃; then (2) enzyme activation and maintenance stage at 95 ℃ for 10 seconds; this was followed by 45 cycles of (3) PCR stage, denaturation at 95 ℃ for 5 seconds, and binding/extension at 60 ℃ for 18 seconds. Thus, the total reaction time for the real-time RT-PCR of the present embodiment is 23 minutes. However, in the case of the commercially available competitive product, the total reaction time is about 77 minutes because reverse transcription requires 20 minutes, enzyme activation requires 2 minutes, and 45 cycles of PCR are added, which is 15 seconds for denaturation, 45 seconds for binding, and 15 seconds for extension.

TABLE 2

In the fast cycle mode of the present embodiment, the following four aspects can save a lot of time. First, the binding/extension time is reduced to 18 seconds to save time, which is typically 60 seconds to counter a commercially available competitive product. Second, the time for denaturation is reduced to 5 seconds to save time, which is usually 15 seconds against commercially available competitive products. Third, reverse transcription time was reduced to 5 minutes, which is typically 20 minutes to counter market competitive products. Fourth, the enzyme activation time is shortened to 10 seconds, which usually takes 2 minutes to counter the competitive products on the market.

Therefore, the time saved by the rapid cycling mode of the primers and probes of the embodiments of the present invention on the same type of thermal cycler can shorten the total reaction time by at least 70% compared to the standard reverse transcription polymerase chain reaction, so the embodiments of the present invention can complete the detection of the retrovirus and the zakhstan virus in less than 30 minutes. In other words, by optimizing the design to reduce the denaturation/binding/extension time of each cycle, reduce the reverse transcription time and reduce the enzyme activation time, the total reaction time is shortened by at least 70%, so that the primers and probes of the present embodiment can achieve rapid multiplex detection of the retrovirus and the zika virus by real-time reverse transcription polymerase chain reaction, and these are all achieved without affecting the sensitivity and specificity of the detection of the retrovirus and the zika virus.

Examples of multiplex assays for the Hericium virus and the Zika virus of the present embodiment will be described below, wherein the reaction mixtures are shown in Table 3. The real-time reverse transcription polymerase chain reaction was performed using the rapid cycling protocol described above, and the reaction mixture included One Step PrimeScript from Takara^TMRT-PCR kit (Perfect Real Time; # RR064A), 500nM forward primer (SEQ ID NO:1), 500nM reverse primer (SEQ ID NO:2), 200nM probe (SEQ ID NO:3), 500nM forward primer (SEQ ID NO:4), 500nM forward primer (SEQ ID NO:5), 500nM reverse primer (SEQ ID NO:6), 200nM probe (SEQ ID NO:7), 250nM forward primer (SEQ ID NO:8), 250nM reverse primer (SEQ ID NO:9), 100nM probe (SEQ ID NO:10), 1000 copies of proA internal control IVT-RNA, and appropriate amounts of RNA extract samples in a total volume of 20. mu.L. However, the reaction mixture is not limited to the above-mentioned enzymes and buffer systems.

TABLE 3

To further experimentally confirm the specificity of primers and probes for the retrovirus and zirca viruses in multiplex assays, the primers and probes were tested for cross-reactivity with several common mosquito-borne viruses, including Dengue virus first to fourth serotypes 1 to 4, in large amounts of genomic RNA (approximately between 10A)⁶To 10⁷Copied gRNA) was performed in the presence of a dna template. FIGS. 4 and 5 show the results of the analysis of the specificity of the virus for drogonella virus and Zika virus, respectively, wherein CHIK, DEN1 to DEN4, and ZIKA represent genomic RNA samples from the virus for drogonella virus, the first to fourth serotypes of dengue virus, and the virus for Zika, respectively, and NTC represents a template-free control group. In the fluorescence channel for the detection of the Cherov virus, as shown in FIG. 4, no amplification reaction was observed in any of the groups except for the amplification reaction in the Cherov genomic RNA sample. Similarly, in the fluorescent channel for the detection of the Zerca virus, as shown in FIG. 5, except that the Zerca genomic RNA sample has amplification reactionIn addition, no amplification reaction was observed in any of the other groups. In other words, no cross reaction occurs in this experiment, thus confirming the specificity of the primers and probes in the multiplex detection.

The embodiment of the invention can detect the drogong genome RNA with only a plurality of copies per reaction in a multiplex detection within less than 30 minutes. Reactions containing 1, 3, 5, 8, 10 and 15 copies of the genomic dna of drogonella were analyzed for 6 replicates, respectively, and the threshold for positive detection was set as Ct 38. The results showed that the positive detection rates for reactions containing 1, 3, 5, 8, 10 and 15 copies per reaction were 3/6, 5/6, 5/6, 6/6, 6/6 and 6/6, respectively. In other words, drometrivirus was detected in 6 replicates in reactions containing 8, 10 and 15 copies per reaction. Therefore, the bottom line of detection (LoD) of the drogonella virus in the multiplex detection can be predicted to be about 8 to 10 copies, showing a considerably high sensitivity. In addition, the bottom line of detection was confirmed by repeating 20 analyses of reactions containing 10 copies of the genomic RNA per reaction, and all the 20 repeats resulted in positive detection results, so that the bottom line of detection was confirmed to be 10 copies per reaction (the positive detection rate based on 20/20 was more than 95%).

The present embodiment also enables detection of only a few copies of genomic RNA of the Zrca per reaction in a multiplex assay in less than 30 minutes. Reactions containing 1, 3, 5, 8, 10 and 15 copies of genomic RNA of the Zka were analyzed 6 replicates each, and the cut-off for positive detection was set as Ct value 38. The results showed that the positive detection rates for reactions containing 1, 3, 5, 8, 10 and 15 copies per reaction were 3/6, 2/6, 5/6, 3/6, 6/6 and 6/6, respectively. In other words, zirca virus was detected in 6 replicates in reactions containing 10 and 15 copies per reaction. Therefore, the bottom line of detection (LoD) of the zirca virus in the multiplex detection can be predicted to be about 10 to 15 copies, showing a considerably high sensitivity. In addition, the bottom line of detection was confirmed by further performing 20 replicates of reactions containing 10 copies of genomic RNA of the Zukka gene per reaction, and as a result, 19 reactions out of the 20 replicates gave a positive detection result, so that the bottom line of detection was confirmed to be 10 copies per reaction (the positive detection rate based on 19/20 was 95% reliable).

The embodiments of the present invention can detect both Cherovirus and Zika virus in the same real-time RT-PCR. Two reactions were analyzed in a multiplex assay, one reaction having 100 copies of the genomic DNA and 100 copies of the genomic DNA of Z-Cara, and the other reaction having 10 copies of the genomic DNA and 10 copies of the genomic RNA of Z-Cara. FIGS. 6A and 6B show the results of joint detection of the Tourette virus and the Zika virus. FIGS. 6A and 6B show that when 100 copies of the genomic DNA of Cherokee virus and 100 copies of the genomic RNA of Zika virus were simultaneously present in the reaction, the multiple detection could detect both the Cherokee virus and the Zika virus. When 10 copies of the Pythagorean genomic RNA and 10 copies of the Tzka genomic RNA are simultaneously present in the reaction, the multiplex assay detects the Pythagorean virus in all of the 2 replicate assays and the Tzka virus in one of the 2 replicate assays. Thus, multiplex assays can simultaneously detect drogonvirus and zirca virus even if the viral RNA content is low.

The embodiment of the scheme can quantify the droutherous genome RNA in less than 30 minutes. FIG. 7 shows amplification curves generated from different copy numbers of drogue genomic RNA. Using the primers and probes of the present examples, there were serially diluted copy numbers per reaction (10)⁷To 10 copies) of the drogue genomic RNA sample were tested separately to determine the linearity of the drogue assay in the multiplex assay. FIG. 8 shows the flexor analysis standard curve resulting from the results of FIG. 7. According to the obtained standard curve, the amplification efficiency was 102%, R²The value was 1.00. Due to R²The optimal value is 1.0, so the multiplex assay can be extended to quantitative analysis to estimate the amount of genomic RNA in the POC assay.

The embodiment can also quantify the genomic RNA of the Zka in less than 30 minutes. FIG. 9 shows amplification curves generated from genomic RNAs of Zirca virus at different copy numbers. The primers and probes of the embodiments are used and have continuous dilution for each reactionCopy number (2x 10)⁶To 20 copies) of the genomic RNA sample of zika were tested separately to determine the linearity of the zika detection in a multiplex assay. FIG. 10 shows a standard curve of the Zercard analysis generated from the results of FIG. 9. According to the obtained standard curve, the amplification efficiency was 104%, R²The value was 1.00. Due to R²The optimal value is 1.0, so the multiplex assay can be extended to quantitative analysis to estimate the amount of genomic RNA of the zica at the time of POC detection.

The multiplex assays for the Trigonella virus and the Zika virus of the present example were further compared with two commercially available Trigonella assay kits or two commercially available Zika assay kits. The comparative experiments were performed with small amounts of viral RNA, e.g., 10 or 100 copies of genomic RNA per reaction. FIG. 11 shows the comparison of the multiplex assay of the present invention with two commercially available Trigonella assay kits, and FIG. 12 shows the comparison of the multiplex assay of the present invention with two commercially available Zehnder assay kits.

As shown in fig. 11, when a small amount of drogue RNA was added to the reaction, the average Ct values of the multiplex assay, the commercial kit 1, and the commercial kit 2 were 31.673, 34.055, and 33.196, respectively, for the 100-copy genomic RNA assay; for the detection of 10 copies of genomic RNA, the average Ct values of the multiplex detection, the commercial kit 1 and the commercial kit 2 are 34.415, 37.617 and 36.461, respectively. Therefore, the multiple detection of the scheme can detect the drogonella virus by the early Ct, so the multiple detection of the scheme is better than that of two commercially available kits when a small amount of drogonella RNA is detected. In addition, the total reaction time of the real-time reverse transcription polymerase chain reaction of the multiplex assay, the commercial kit 1 and the commercial kit 2 is 23 minutes, 77 minutes and 62 minutes, respectively, so the multiplex assay takes a significantly shorter time to complete the real-time reverse transcription polymerase chain reaction compared to the commercial kit.

As shown in fig. 12, when a small amount of zaka RNA was added to the reaction, the average Ct values of the multiplex assay, the commercial kit 1, and the commercial kit 2 in this case were 31.953, 33.711, and 32.101, respectively, for the detection of 100 copies of genomic RNA; for the detection of 10 copies of genomic RNA, the average Ct values of the multiplex detection, the commercial kit 1 and the commercial kit 2 are 35.983, 36.304 and 35.480, respectively. Therefore, the multiplex detection can detect the Zka virus by early Ct or comparable Ct, so that the multiplex detection is better than or comparable to two commercial kits when detecting a small amount of the Zka RNA. In addition, the total reaction time of the real-time reverse transcription polymerase chain reaction of the multiplex assay, the commercial kit 1 and the commercial kit 2 is 23 minutes, 77 minutes and 62 minutes, respectively, so the multiplex assay takes a significantly shorter time to complete the real-time reverse transcription polymerase chain reaction compared to the commercial kit.

In summary, the embodiments of the present disclosure provide a multiplex detection kit and method for a virus of the Torpedo virus and a virus of the Torpedo virus, which utilize real-time reverse transcription polymerase chain reaction and specific primers and probes for detection. The multiplex detection kit and method of the present embodiment have the advantage of detecting the Tourette virus and the Zika virus simultaneously with high sensitivity and high specificity. The multiplex assay kits and methods of the present embodiments can detect low copy number of Pythagorean and Zucuca RNA and can be used for quantitative detection. The multiplex assay kit and method of the present embodiment further have the advantage of reducing the reaction time, and are realized without affecting the sensitivity and specificity. Thus, the present disclosure provides a rapid, sensitive, specific, and multiplex assay that can simultaneously detect the drogonella virus and the zika virus, thereby effectively treating infection and preventing transmission.

While the present invention has been described in detail with respect to the above embodiments, it will be apparent to those skilled in the art that various modifications can be made without departing from the scope of the invention as defined in the appended claims.

Sequence listing

<110> Taida Electronics International (Singapore) private company, Inc. (Delta Electronics Int ˇ l (Singapore) Pte Ltd)

<120> multiple detection kit and method for drogong virus and zika virus

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<170> PatentIn version 3.5

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Claims

1. A multiple detection kit for a virus of Torpedo virus and a virus of Zika virus comprises at least one of a first primer group specific to the virus of Torpedo virus and a second primer group specific to the virus of Zika virus,

wherein the first primer set is selected from:

(a) a forward primer having the nucleotide sequence of SEQ ID NO. 1 and a reverse primer having the nucleotide sequence of SEQ ID NO. 2; and

(b) a forward primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 1 and a reverse primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 2,

wherein the second primer set is selected from:

(a) a forward primer having the nucleotide sequence of SEQ ID NO. 4 and a reverse primer having the nucleotide sequence of SEQ ID NO. 6;

(b) a forward primer having the nucleotide sequence of SEQ ID NO. 5 and a reverse primer having the nucleotide sequence of SEQ ID NO. 6;

(c) a first forward primer having the nucleotide sequence of SEQ ID NO. 4, a second forward primer having the nucleotide sequence of SEQ ID NO. 5, and a reverse primer having the nucleotide sequence of SEQ ID NO. 6; and

(d) at least one forward primer having a nucleotide sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO. 4 and SEQ ID NO. 5, and a reverse primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 6.

2. The multiplex assay kit of claim 1 further comprising a probe specific for droguet virus, wherein the probe has a nucleotide sequence selected from the group consisting of:

(a) 3, the nucleotide sequence of SEQ ID NO;

(b) a nucleotide sequence complementary to SEQ ID NO. 3; and

(c) a nucleotide sequence having at least 90% identity to SEQ ID NO. 3 or the complement thereof.

3. The multiplex assay kit of claim 2, wherein the probe is 5 'terminated with a reporter dye and 3' terminated with a quencher.

4. The multiplex assay kit of claim 1 further comprising a probe specific for zirca virus, wherein the probe has a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO. 7;

(b) a nucleotide sequence complementary to SEQ ID NO. 7; and

(c) a nucleotide sequence having at least 90% identity to SEQ ID NO 7 or the complement thereof.

5. The multiplex assay kit of claim 4, wherein the probe is 5 'terminated with a reporter dye and 3' terminated with a quencher.

6. The multiplex assay kit of claim 1 further comprising an internal control template which is RNA transcribed in vitro from the proA gene of a soft-rot bacterium (pevibacterium carotovorum).

7. The multiplex assay kit of claim 6, further comprising a control primer set specific for the proA gene, wherein the control primer set is selected from the group consisting of:

(a) a forward primer having the nucleotide sequence of SEQ ID NO. 8 and a reverse primer having the nucleotide sequence of SEQ ID NO. 9; and

(b) a forward primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 8 and a reverse primer having a nucleotide sequence at least 90% identical to SEQ ID NO. 9.

8. The multiplex assay kit of claim 7 further comprising a probe specific for the proA gene, wherein the probe has a nucleotide sequence selected from the group consisting of:

(a) 10, the nucleotide sequence of SEQ ID NO;

(b) a nucleotide sequence complementary to SEQ ID NO. 10; and

(c) a nucleotide sequence having at least 90% identity to SEQ ID NO 10 or its complement.

9. The multiplex assay kit of claim 8, wherein the probe is 5 'terminated with a reporter dye and 3' terminated with a quencher.

10. A multiple detection method for a virus of drogong and a virus of zika comprises the following steps:

(a) contacting a biological sample with the multiplex assay kit of claim 1;

(b) amplifying nucleic acids of the virus and/or the zirca virus from the biological sample; and

(c) detecting the presence of amplified nucleic acids of the virus of Tourette's and/or Zika's virus.

11. The multiplex assay of claim 10 further comprising a step of extracting nucleic acids from the biological sample prior to step (a).

12. The multiplex assay of claim 10 wherein step (b) comprises the substeps of:

(b1) reverse transcribing viral genomic RNA to complementary DNA; and

(b2) the complementary DNA is amplified using a real-time polymerase chain reaction.

13. The multiplex assay of claim 10 wherein the multiplex assay kit further comprises a first probe specific for the Tohrysvirus and/or a second probe specific for the Zircavirus,

wherein the first probe has a nucleotide sequence selected from the group consisting of:

(a) 3, the nucleotide sequence of SEQ ID NO;

(b) a nucleotide sequence complementary to SEQ ID NO. 3; and

(c) a nucleotide sequence having at least 90% identity to SEQ ID NO 3 or the complement thereof,

wherein the second probe has a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO. 7;

(b) a nucleotide sequence complementary to SEQ ID NO. 7; and

14. The multiplex assay of claim 13 wherein the first probe and the second probe are both conjugated to a reporter dye at the 5 'terminus and a quencher at the 3' terminus.

15. The multiplex assay of claim 14 wherein the 5' termini of the first probe and the second probe are attached to different reporter dyes.

16. The multiplex assay of claim 10 wherein the multiplex assay kit further comprises an internal control template which is RNA transcribed in vitro from the proA gene of a soft-rot bacterium (pebacter carotovorum).

17. The multiplex assay of claim 16 wherein the multiplex assay kit further comprises a control primer set specific for the proA gene, wherein the control primer set is selected from the group consisting of:

18. The multiplex assay of claim 17 wherein the multiplex assay kit further comprises a probe specific for the proA gene, wherein the probe has a nucleotide sequence selected from the group consisting of seq id nos:

(a) 10, the nucleotide sequence of SEQ ID NO;

(b) a nucleotide sequence complementary to SEQ ID NO. 10; and

19. The multiplex assay of claim 18 wherein the probe is 5 'terminated with a reporter dye and 3' terminated with a quencher.