LAMP (loop-mediated isothermal amplification) primer combination for detecting human parainfluenza virus type 4, kit and application

Abstract

The invention relates to the technical field of biology, in particular to an LAMP primer combination for detecting human parainfluenza virus type 4, a kit and application. The LAMP primer combination for detecting the human parainfluenza virus 4 provided by the invention comprises the following components: a primer pair, a primer pair and a primer pair, wherein the primer pair is designed on the basis of an HN gene of the human parainfluenza virus 4; the LAMP primer combination designed on the basis of the HN gene of the human parainfluenza virus 4 can detect the human parainfluenza virus 4 in a high-sensitivity and high-specificity mode, rapid detection can be achieved in a short time, and an efficient tool is provided for clinical diagnosis and epidemiological monitoring of the human parainfluenza virus 4.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a LAMP primer combination, a kit and uses for detecting human parainfluenza virus type 4. Background Art

[0002] Human Parainfluenza Virus 4 (HPIV-4) is a member of the Paramyxoviridae genus. Although it has been relatively poorly studied clinically, it still plays a certain role in respiratory infections. HPIV-4 infection mainly causes mild upper respiratory tract symptoms such as cough, runny nose and sore throat, usually manifesting as a common cold. However, in infants, the elderly and immunosuppressed people, HPIV-4 infection may cause more severe lower respiratory tract diseases such as bronchitis and pneumonia, and even require hospitalization. Studies have shown that the detection rate of HPIV-4 in acute respiratory tract infections in children is approximately 1%-5%. Although it is lower than other parainfluenza viruses (such as HPIV-1 and HPIV-3), its potential clinical impact cannot be ignored.

[0003] HPIV-4 prevalence is seasonal, typically peaking in the fall and winter. This may overlap with the prevalence of other respiratory viruses, increasing the risk of co-infection. Furthermore, because HPIV-4 detection methods are relatively limited and its symptoms are similar to those of other respiratory viral infections, missed or misdiagnosed cases may occur, thereby underestimating its actual clinical burden. In immunosuppressed patients (such as organ transplant recipients or those infected with HIV), HPIV-4 infection can cause serious complications and even be life-threatening.

[0004] Although the clinical significance of HPIV-4 is not yet fully understood, its potential impact as a respiratory pathogen warrants further investigation. Developing more sensitive and specific detection methods and strengthening epidemiological surveillance are crucial to fully understand the clinical significance of HPIV-4 and develop effective prevention and control strategies.

[0005] Loop-mediated isothermal amplification (LAMP) is an efficient, rapid, and specific nucleic acid amplification technique. LAMP utilizes a DNA polymerase with strand displacement activity (such as BstDNA polymerase) and a set of specifically designed primers to exponentially amplify the target DNA sequence under constant temperature conditions (typically 60-65°C). Unlike traditional polymerase chain reaction (PCR), LAMP requires only a constant temperature heater and does not require a thermal cycler, making it particularly suitable for resource-limited point-of-care testing (POCT) or point-of-care testing. LAMP primers typically include two outer primers (F3 and B3), two inner primers (FIP and BIP), and optional two loop primers (LF and LB). These primers recognize multiple specific regions within the target DNA sequence, ensuring high specificity and sensitivity. The LAMP reaction products are a series of DNA fragments of varying lengths and structures, typically interpreted using turbidity detection, fluorescent dyes, or colorimetric assays. Due to its simple and rapid operation (usually completed within 30-60 minutes) and low equipment requirements, LAMP technology has been widely used in pathogen detection, food safety monitoring, environmental microbial analysis, and genetic disease diagnosis.

[0006] However, there are relatively few documents that utilize loop-mediated isothermal amplification (LAMP) technology to detect human parainfluenza virus type 4 (HPIV-4). For example, patent document CN119161973A only discloses the use of LAMP technology to detect human parainfluenza virus, but does not specifically detect human parainfluenza virus subtypes (such as HPIV-1, HPIV-2, HPIV-3, and HPIV-4). The reason why loop-mediated isothermal amplification (LAMP) technology is relatively rare for detecting human parainfluenza virus type 4 (HPIV-4) may be that the genome variability of HPIV-4 is high, especially mutations in the fusion protein (F) and hemagglutinin-neuraminidase (HN) genes may cause changes in primer binding sites, thereby reducing the sensitivity and specificity of LAMP detection. LAMP primer design requires recognition of 6-8 specific regions on the target sequence, which places high demands on the selection of highly conserved regions in the HPIV-4 genome and is relatively difficult to design. Furthermore, HPIV-4 shares a high degree of genomic sequence homology with other parainfluenza viruses (such as HPIV-1 and HPIV-3), which may cause primers to cross-react with non-target viruses, resulting in false-positive results. Furthermore, existing LAMP primer design tools (such as PrimerExplorer) may not fully account for sequence differences between viral strains when designing for the HPIV-4 genome, resulting in unstable primers in practical applications. Furthermore, the presence of inhibitors in HPIV-4 samples (such as mucus or cellular debris in respiratory secretions) may further affect the efficiency and accuracy of the LAMP reaction. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to provide a LAMP primer combination, a kit and a use for detecting human parainfluenza virus type 4, which can detect human parainfluenza virus type 4 with high sensitivity and high specificity, and can be quickly detected in a short time, providing an efficient tool for the clinical diagnosis and epidemiological monitoring of human parainfluenza virus type 4.

[0008] To this end, the present invention provides the following technical solutions:

[0009] Embodiments of the present invention provide a LAMP primer combination for detecting human parainfluenza virus type 4, comprising primers designed based on bp 2 to 1240 of the HN gene of human parainfluenza virus type 4. The present invention has found that designing a LAMP primer combination based on this conserved region enables high sensitivity and specificity in detecting human parainfluenza virus type 4, and enables rapid detection within a short period of time.

[0010] In some embodiments, the LAMP primer combination for detecting human parainfluenza virus type 4 comprises:

[0011] Outer primer set:

[0012] Outer primer F3: nucleotide sequence as shown in SEQ ID NO.1;

[0013] Outer primer B3: nucleotide sequence as shown in SEQ ID NO.2;

[0014] Inner primer set:

[0015] Internal primer FIP: nucleotide sequence is shown in SEQ ID NO.3;

[0016] Internal primer BIP: nucleotide sequence is shown in SEQ ID NO.4;

[0017] Loop primer set:

[0018] Loop primer LF: nucleotide sequence is shown in SEQ ID NO.5;

[0019] Loop primer LP: nucleotide sequence is shown in SEQ ID NO.6;

[0020] or

[0021] Outer primer set:

[0022] Outer primer F3: nucleotide sequence is shown in SEQ ID NO.7;

[0023] Outer primer B3: nucleotide sequence is shown in SEQ ID NO.8;

[0024] Inner primer set:

[0025] Internal primer FIP: nucleotide sequence is shown in SEQ ID NO.9;

[0026] Internal primer BIP: nucleotide sequence is shown in SEQ ID NO.10;

[0027] Loop primer set:

[0028] Loop primer LF: nucleotide sequence is shown in SEQ ID NO.11;

[0029] Loop primer LP: The nucleotide sequence is shown in SEQ ID NO.12.

[0030] In some embodiments, the molar ratio of the final concentration of the outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF, and loop primer LP is 0.2-0.5 μM: 0.2-0.5 μM: 1-2 μM: 1-2 μM: 0.4-0.5 μM: 0.4-0.5 μM. The molar ratio of the final concentration of the system of the outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF, and loop primer LP can be any one of 0.2 μM: 0.5 μM: 1 μM: 2 μM: 0.4 μM: 0.5 μM, 0.5 μM: 0.2 μM: 2 μM: 1 μM: 0.5 μM: 0.4 μM, 0.25 μM: 0.25 μM: 1.5 μM: 1.5 μM: 0.45 μM: 0.45 μM or 0.2 μM: 0.2 μM: 1.6 μM: 1.6 μM: 0.4 μM: 0.4 μM, or a range value between any two values.

[0031] An embodiment of the present invention provides use of the LAMP primer combination for detecting human parainfluenza virus type 4 in preparing a product for detecting human parainfluenza virus type 4.

[0032] In some embodiments, the product comprises a reagent, a kit, or a chip.

[0033] An embodiment of the present invention provides a kit for detecting human parainfluenza virus type 4, comprising the LAMP primer combination for detecting human parainfluenza virus type 4.

[0034] In some embodiments, a loop-mediated isothermal amplification reaction system is further included; the isothermal amplification reaction system includes Bst DNA polymerase, reverse transcriptase, fluorescent dye, buffer and / or dNTP.

[0035] In some embodiments, the loop-mediated isothermal amplification reaction system:

[0036] Bst Buffer: 5-7 μL;

[0037] 10× primer mix: 2-3 μL;

[0038] Bst Enzyme Mix (UDG plus): 0.5~1.5μL;

[0039] Template: concentration 25-100 copies / reaction, 1-3 μL;

[0040] Enzyme-free water: make up to 25 μL;

[0041] In the loop-mediated isothermal amplification reaction system, the molar ratio of the final concentration of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF, and the loop primer LP is 0.2-0.5 μM: 0.2-0.5 μM: 1-2 μM: 1-2 μM: 0.4-0.5 μM: 0.4-0.5 μM.

[0042] In some preferred embodiments, the loop-mediated isothermal amplification reaction system:

[0043] Bst Buffer: 6 μL;

[0044] 10× primer mix: 2.5 μL;

[0045] Bst Enzyme Mix (UDG plus): 1μL;

[0046] Template: concentration 25-100 copies / reaction, 2 μL;

[0047] Enzyme-free water: make up to 25 μL.

[0048] The Bst Buffer is a commercially available product, containing a buffer, dNTPs, and the fluorescent dye required for detection. The Bst Enzyme Mix (UDG plus) is a commercially available product, a mixed enzyme solution including Bst DNA polymerase and reverse transcriptase.

[0049] An embodiment of the present invention provides a method for detecting human parainfluenza virus type 4, comprising the following steps:

[0050] S1, extracting RNA from the sample to be tested;

[0051] S2. Using the LAMP primer combination for detecting human parainfluenza virus type 4 or the kit for detecting human parainfluenza virus type 1, a loop-mediated isothermal amplification reaction is performed using the cDNA in step S1 as a template;

[0052] S3. After the loop-mediated isothermal amplification reaction is completed, determine whether an amplification product is amplified in the loop-mediated isothermal amplification reaction system, and determine whether the sample to be tested contains human parainfluenza virus type 4.

[0053] In some embodiments, the conditions of the loop-mediated isothermal amplification reaction are: incubation at 37°C for 2-5 minutes; incubation at a constant temperature of 60-65°C for 30-60 minutes; inactivation at 85°C for 4-6 minutes. In the loop-mediated isothermal amplification reaction, the purpose of incubation at 37°C for 2-5 minutes is to activate the activity of the UDG enzyme and prevent the impact of aerosol pollution in the environment on expansion. In some embodiments, the incubation is at 37°C for 2 minutes, 3 minutes, 4 minutes or 5 minutes. In some embodiments, the incubation is at a constant temperature of 60-65°C for 30-60 minutes, such as any one of 60, 61, 62, 63, 64, and 65°C or a range between any two values, and the incubation time can be any one of 30, 35, 40, 45, 50, and 60 minutes or a range between any two values. A preferred embodiment is incubation for 30 minutes.

[0054] The technical solution of the present invention has the following advantages:

[0055] 1. The present invention provides a LAMP primer combination for detecting human parainfluenza virus type 4, comprising: a primer designed based on the 2nd to 1240th base pair of the HN gene of human parainfluenza virus type 4; the present invention screens out highly conserved gene regions of human parainfluenza virus type 4 as targets through extensive screening and comparison, wherein the gene regions are selected from the nucleocapsid protein gene (NP), fusion protein gene (F), toxin gene (yruI / yruR), or 16S rRNA gene. After further screening and verification, it was found that the HN gene of HPIV4 belongs to HPIV4 type A and is in an independent branch in terms of taxonomic status, indicating that the HN gene of HPIV4 has good specificity and is universal in HPIV4; therefore, the LAMP primer combination designed based on the HN gene of human parainfluenza virus type 4 can detect human parainfluenza virus type 4 with high sensitivity and specificity, and can be rapidly detected in a short time, providing an efficient tool for clinical diagnosis and epidemiological monitoring of human parainfluenza virus type 4.

[0056] 2. The present invention provides a LAMP primer combination for detecting human parainfluenza virus type 4, comprising: an outer primer group: an outer primer F3: a nucleotide sequence as shown in SEQ ID NO.1; an outer primer B3: a nucleotide sequence as shown in SEQ ID NO.2; an inner primer group: an inner primer FIP: a nucleotide sequence as shown in SEQ ID NO.3; an inner primer BIP: a nucleotide sequence as shown in SEQ ID NO.4; a loop primer group: a loop primer LF: a nucleotide sequence as shown in SEQ ID NO.5; a loop primer LP: a nucleotide sequence as shown in SEQ ID NO.6; or an outer primer group: an outer primer F3: a nucleotide sequence as shown in SEQ ID NO.7; an outer primer B3: a nucleotide sequence as shown in SEQ ID NO.8; an inner primer group: an inner primer FIP: a nucleotide sequence as shown in SEQ ID NO.9; an inner primer BIP: a nucleotide sequence as shown in SEQ ID NO.10; a loop primer group: a loop primer LF: a nucleotide sequence as shown in SEQ ID NO.11; a loop primer LP: a nucleotide sequence as shown in SEQ ID NO. As shown in NO.12; the LAMP primer combination designed in the present invention can detect human parainfluenza virus type 4 with high sensitivity and high specificity, and can quickly detect in a short time, with the highest sensitivity reaching 6.25 copies / reaction, a detection rate of 95%, and rapid detection within 30 minutes, providing an efficient tool for clinical diagnosis and epidemiological monitoring of human parainfluenza virus type 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0058] Figure 1 is the position of primer set No. 1 in Example 1 of the present invention in the HN gene of HPIV4;

[0059] Figure 2 is the position of primer set No. 2 in Example 1 of the present invention in the HN gene of HPIV4;

[0060] Figure 3 This is the primer wet method verification result in Experimental Example 1 of the present invention;

[0061] Figure 4 is the sensitivity test result of primer set 1 in Experimental Example 2 of the present invention;

[0062] Figure 5is the sensitivity test result of primer set 2 in Experimental Example 2 of the present invention;

[0063] Figure 6 The results of the primer set 1 in Experimental Example 2 of the present invention for 20 groups of samples are shown below:

[0064] Figure 7 This is the specific detection result of primer set 1 in Experimental Example 3 of the present invention.

[0065] Figure 8 This is the clinical sample detection result of primer set 1 in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0066] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0067] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0068] Bst Buffer and Bst Enzyme Mix (UDG plus) were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0069] Example 1 Target gene screening and primer design

[0070] This embodiment screened out highly conserved gene regions of human parainfluenza virus type 4 as targets through a large amount of screening and comparison. The gene regions were selected from the nucleocapsid protein gene (NP), fusion protein gene (F), toxin gene (yruI / yruR) or 16S rRNA gene. After further screening and verification, it was found that the HN gene of HPIV4 belongs to type A of HPIV4 and is in an independent branch in terms of classification status, indicating that the HN gene of HPIV4 has good specificity and is universal in HPIV4. Therefore, this embodiment designed primers based on the HN gene of HPIV4, and the primers were synthesized by Sangon Biotech Co., Ltd. The primer sequences are shown in Table 1 below. The positions of the primers in the HN gene of HPIV4 are as follows: Figure 1-Figure 2 shown

[0071] Table 1: HPIV4 primer design sequence list

[0072]

[0073]

[0074] Example 2 Kit for detecting human parainfluenza virus type 4

[0075] This embodiment provides a kit for detecting human parainfluenza virus type 4, comprising any one of the primer sets in Example 1.

[0076] Furthermore, it also includes a loop-mediated isothermal amplification reaction system; the isothermal amplification reaction system includes Bst DNA polymerase, buffer and / or dNTP.

[0077] Furthermore, the loop-mediated isothermal amplification reaction system is as follows:

[0078] Bst Buffer (containing dNTPs and SYTO-9 fluorescent dye, etc.): 6 μL;

[0079] 10× primer mix: 2.5 μL;

[0080] Bst Enzyme Mix (UDG plus): 1μL;

[0081] Template: concentration 25-100 copies / reaction, 2 μL;

[0082] Enzyme-free water: make up to 25 μL;

[0083] In the loop-mediated isothermal amplification reaction system, the final concentration of the outer primer F3 is 0.2 μM, the final concentration of the outer primer B3 is 0.2 μM, the final concentration of the inner primer FIP is 1.6 μM, the final concentration of the inner primer BIP is 1.6 μM, the final concentration of the loop primer LF is 0.4 μM, and the final concentration of the loop primer LP is 0.4 μM.

[0084] Example 3 Method for Detecting Human Parainfluenza Virus Type 4

[0085] This embodiment provides a method for detecting human parainfluenza virus type 4, comprising the following steps:

[0086] S1, extracting RNA from the sample to be tested;

[0087] The RNA template of HPIV4 in this example was purchased from Twiss nucleic acid standard (original concentration: 10 6 copies / μL)

[0088] S2. Using the kit for detecting human parainfluenza virus type 4 described in Example 2, a loop-mediated isothermal amplification reaction was performed using the cDNA in step S1 as a template; the prepared loop-mediated isothermal amplification reaction system was as follows:

[0089] Bst Buffer: 6 μL;

[0090] 10× primer mix: 2.5 μL;

[0091] Bst Enzyme Mix (UDG plus): 1μL;

[0092] Template: 25-100 copies / reaction, 2 μL;

[0093] Enzyme-free water: make up to 25 μL.

[0094] In the loop-mediated isothermal amplification reaction system, the final concentration of the outer primer F3 is 0.2 μM, the final concentration of the outer primer B3 is 0.2 μM, the final concentration of the inner primer FIP is 1.6 μM, the final concentration of the inner primer BIP is 1.6 μM, the final concentration of the loop primer LF is 0.4 μM, and the final concentration of the loop primer LP is 0.4 μM.

[0095] The conditions of the loop-mediated isothermal amplification reaction are: incubation at 37°C for 2 minutes (the reagent used contains the anti-pollution component UDG enzyme, which is activated at 37°C); incubation at a constant temperature of 63°C for 30 minutes; and inactivation at 85°C for 5 minutes.

[0096] From the start of the loop-mediated isothermal amplification reaction to the end of the reaction procedure, the QuantStudio1Plus real-time fluorescence quantitative PCR instrument is used to detect the fluorescence signal in the loop-mediated isothermal amplification reaction system. The positive time (min) is used as the horizontal axis (the positive time is the time point when the fluorescence signal of the positive group sample changes dramatically), and the change in fluorescence intensity (△Rn) is used as the vertical axis to draw a fluorescence amplification curve. The results are judged as follows: the negative group (the sample group containing no nucleic acid, or called the blank group) requires no reaction time, or the reaction time is greater than 27 minutes; the positive amplification group: the reaction time is required to be less than 10 minutes when the template concentration is 100 copies / reaction, which is considered positive; otherwise, it is judged as negative, or the reaction time below 100 copies / reaction is less than 15 minutes, which is considered positive, otherwise it is judged as negative.

[0097] Experimental Example 1 Primer wet method verification

[0098] Primers No. 1 and No. 2 in Table 1 were used to detect human parainfluenza virus type 4, respectively. The method was implemented according to Example 3, except that in the loop-mediated isothermal amplification reaction system, primers No. 1 or No. 2 were selected. Template: HPIV4 RNA template purchased from Twiss nucleic acid standard (original concentration: 10 6The blank group is an amplification group without nucleic acid sample, in which the missing liquid volume is replaced with enzyme-free water (except for the absence of nucleic acid; the remaining amplification components are identical to those of the positive amplification group).

[0099] The results are as follows Figure 3 As shown, the amplification performance of the primer sets was determined by fluorescence signals. The amplification performance of the primers was tested at a template concentration of 100 copies / reaction per reaction. At the same template concentration, primer sets 1 and 2 achieved excellent amplification performance, as evidenced by the positive time for amplification in all positive groups being within 10 minutes, and no fluorescence change in the blank group. This demonstrated good consistency between the positive groups.

[0100] Experimental Example 2 Sensitivity Test

[0101] Primers No. 1 and No. 2 in Table 1 were used to detect human parainfluenza virus type 4, respectively. The method was implemented according to Example 3, except that in the loop-mediated isothermal amplification reaction system, primers No. 1 or No. 2 were selected. Template: HPIV4 RNA template purchased from Twiss nucleic acid standard (original concentration: 10 6 Copies / μL) were serially diluted to concentrations of 200, 100, 50, 25, 12.5, and 6.25 copies / Reaciton, respectively.

[0102] The test results corresponding to primer set 1 are as follows Figure 4 As shown in the figure, the minimum detection limit is 6.25 copies / reaciton.

[0103] The detection results corresponding to primer set 2 are as follows Figure 5 As shown in the figure, the minimum detection limit is 25 copies / reaciton.

[0104] Primer No. 1 in Table 1 was used to detect human parainfluenza virus type 4. The method was implemented according to Example 3. 20 groups of samples were tested: the concentration of HPIV4 RNA template (nucleic acid standard purchased from Twis) was 6.25 copies / reaciton. The results are as follows. Figure 6 As shown, primer set 1 detected 19 of the 20 RNA samples, with a detection rate of 95%. Furthermore, there was no peak in the blank group, indicating that the primer set had good amplification efficiency and no nonspecific amplification.

[0105] Experimental Example 3 Specificity Verification

[0106] Primer Set 1 in Table 1 was used to detect human parainfluenza virus type 4, influenza A virus H3N2, and respiratory syncytial virus (RSV). The assays were performed as in Example 3. RNA templates for human parainfluenza virus type 4, influenza A virus H3N2, and respiratory syncytial virus (RSV) were purchased from Twiss nucleic acid standards and diluted to 100 copies / reaction. Three replicates were performed for each virus.

[0107] The results are as follows Figure 7 As shown, it was found that primer set 1 could not amplify under influenza A virus H3N2 and respiratory syncytial virus RSV, but could only amplify under the template of HPIV4, with good specificity.

[0108] Experimental Example 4 Clinical Sample Testing

[0109] This experimental example investigated the No. 1 primer set of the present invention, and detected 31 nasopharyngeal swab specimens suspected of human parainfluenza virus type 4 (the method was implemented according to Example 3, and each template was diluted to 100 copies / reaction). Fluorescence quantitative PCR (ChamQ Universal SYBR qPCR Master Mix, a universal high-sensitivity dye-based quantitative PCR detection kit purchased from Nanjing Novozymes Biotechnology Co., Ltd.) was used for verification.

[0110] 31 clinical samples were tested simultaneously using the LAMP method and the fluorescent quantitative PCR method. A heat map was drawn using the sample number and the sample's positive time / Ct value. Darker colors indicate shorter times, and lighter colors indicate longer times. Since the instruments for most blank group samples in LAMP and fluorescent quantitative PCR do not provide positive time / Ct values, the maximum amplification time was used instead for aesthetic purposes. The green color in the LAMP positive group indicates the negative group (blank group); the yellow color in the qPCR positive group indicates the negative group (blank group) (the LAMP method amplification incubation period is 30 minutes, and the fluorescent quantitative PCR method amplification period is 40 minutes, so the negative group colors of the two methods are different).

[0111] The test results are as follows Figure 8 As shown, among the 31 groups of clinical samples, the LAMP method detected 27 groups of samples with a positive rate of 87.1% (the percentage of the number of detected samples to the total number of samples); the fluorescence quantitative PCR detected 25 groups of samples with a positive rate of 80.6%, but the detection time used by the LAMP method was significantly shorter than that of the fluorescence quantitative PCR.

[0112] Note: The reaction time of the LAMP method is 30 minutes, and the determination method is the same as in Example 3. For the fluorescence quantitative PCR amplification, a Ct value of less than 35 minutes is considered a positive result, and a Ct value greater than 35 minutes is considered a negative result.

[0113] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A LAMP primer combination for detecting human parainfluenza virus type 4, characterized in that: include: The design was based on bp 2 to 1240 of the HN gene of human parainfluenza virus type 4.

2. The LAMP primer combination for detecting human parainfluenza virus type 4 according to claim 1, characterized in that include: Outer primer set: Outer primer F3: nucleotide sequence as shown in SEQ ID NO.1; Outer primer B3: nucleotide sequence as shown in SEQ ID NO.2; Inner primer set: Internal primer FIP: nucleotide sequence is shown in SEQ ID NO.3; Internal primer BIP: nucleotide sequence is shown in SEQ ID NO.4; Loop primer set: Loop primer LF: nucleotide sequence is shown in SEQ ID NO.5; Loop primer LP: nucleotide sequence is shown in SEQ ID NO.6; or Outer primer set: Outer primer F3: nucleotide sequence is shown in SEQ ID NO.7; Outer primer B3: nucleotide sequence is shown in SEQ ID NO.8; Inner primer set: Internal primer FIP: nucleotide sequence is shown in SEQ ID NO.9; Internal primer BIP: nucleotide sequence is shown in SEQ ID NO.10; Loop primer set: Loop primer LF: nucleotide sequence is shown in SEQ ID NO.11; Loop primer LP: The nucleotide sequence is shown in SEQ ID NO.

12.

3. The LAMP primer combination for detecting human parainfluenza virus type 4 according to claim 2, characterized in that The molar ratio of the final concentration of the system of outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF, and loop primer LP is 0.2-0.5 μM: 0.2-0.5 μM: 1-2 μM: 1-2 μM: 0.4-0.5 μM: 0.4-0.5 μM.

4. Use of the LAMP primer combination for detecting human parainfluenza virus type 4 according to any one of claims 1 to 3 in preparing a product for detecting human parainfluenza virus type 4.

5. The use according to claim 4, characterized in that The product includes a reagent, a kit or a chip.

6. A kit for detecting human parainfluenza virus type 4, characterized in that A LAMP primer combination for detecting human parainfluenza virus type 4 comprising the method according to any one of claims 1 to 3.

7. The kit for detecting human parainfluenza virus type 4 according to claim 6, characterized in that It also includes a loop-mediated isothermal amplification reaction system; the isothermal amplification reaction system includes Bst DNA polymerase, reverse transcriptase, fluorescent dye, buffer and / or dNTP.

8. The kit for detecting human parainfluenza virus type 4 according to claim 7, characterized in that The loop-mediated isothermal amplification reaction system: Bst Buffer: 5-7 μL; 10× primer mix: 2-3 μL; Bst Enzyme Mix (UDG plus): 0.5~1.5μL; Template: concentration 25-100 copies / reaction, 1-3 μL; Enzyme-free water: make up to 25 μL; In the loop-mediated isothermal amplification reaction system, the molar ratio of the final concentration of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF, and the loop primer LP is 0.2-0.5 μM: 0.2-0.5 μM: 1-2 μM: 1-2 μM: 0.4-0.5 μM: 0.4-0.5 μM.

9. A method for detecting human parainfluenza virus type 4, characterized in that The steps include: S1, extracting RNA from the sample to be tested; S2. Using the LAMP primer combination for detecting human parainfluenza virus type 4 according to any one of claims 1 to 3 or the kit for detecting human parainfluenza virus type 1 according to any one of claims 6 to 8, a loop-mediated isothermal amplification reaction is performed using the cDNA in step S1 as a template; S3. After the loop-mediated isothermal amplification reaction is completed, determine whether an amplification product is amplified in the loop-mediated isothermal amplification reaction system, and determine whether the sample to be tested contains human parainfluenza virus type 4.

10. The method for detecting human parainfluenza virus type 4 according to claim 9, characterized in that: The conditions of the loop-mediated isothermal amplification reaction are: incubation at 37° C. for 2-5 minutes; incubation at a constant temperature of 60-65° C. for 30-60 minutes; and inactivation at 85° C. for 4-6 minutes.

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