Dual fluorescent PCR kit for simultaneous detection of nipah virus type B and nipah virus type M

Abstract

The application discloses a double fluorescence PCR kit for simultaneously detecting B-type and M-type nipah virus, and belongs to the field of nucleic acid detection. The kit comprises a primer mixture composed of a B-type nipah virus specific primer pair and an M-type nipah virus specific primer pair, a non-specific fluorescent dye, an optimized RT-PCR buffer, and positive and negative control products. The kit can be used for simultaneously detecting B-type and M-type nipah virus in one tube, and the precise typing of the two subtypes can be realized through the difference in melting curve Tm values, wherein the Tm value of the M-type nipah virus is 77.0 DEG C, and the Tm value of the B-type nipah virus is 81.5 DEG C. The kit is rapid in reaction, high in sensitivity and strong in specificity, and the detection limit of the B-type and M-type nipah virus is 500 copies / mL. The kit has a good application prospect for early diagnosis of nipah virus caused human-animal shared diseases, port quarantine and epidemiological monitoring.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, and in particular to a dual fluorescent PCR kit for the simultaneous detection of Nipah virus type B and type M. Background Technology

[0002] Nipah virus (NiV) belongs to the genus Hennipah virus in the family Paramyxoviridae. It is an enveloped, single-stranded, negative-sense RNA virus and a highly pathogenic zoonotic virus that poses a serious threat to human and animal health. It has a strong ability to spread from person to person and a case fatality rate as high as 40%-75%. It has been listed by the World Health Organization as a key emerging and re-emerging infectious disease pathogen for prevention and control.

[0003] Based on differences in genomic sequence, Nipah virus is mainly divided into two subtypes: type B (Bangladesh type) and type M (Malaysian type). The two subtypes differ significantly in pathogenicity, transmission routes, and clinical symptoms: type M Nipah virus is mainly characterized by neurological symptoms, and human-to-human transmission is rare. It is mainly mediated by intermediate hosts such as pigs and first broke out in Malaysia in 1998. Type B Nipah virus is mainly characterized by respiratory symptoms and can achieve efficient human-to-human transmission through close contact and droplets. It poses a higher public health risk and has broken out in Bangladesh, India, and other regions.

[0004] Because the clinical symptoms of Nipah virus types B and M overlap, and there are currently no specific treatments or commercial vaccines, early rapid detection and accurate typing are crucial for the prevention and control of Nipah virus. Existing Nipah virus detection methods mainly include single-type fluorescent PCR detection, serological testing, and gene sequencing. Serological testing suffers from long window periods and low sensitivity, while gene sequencing is time-consuming and costly, making it unsuitable for rapid on-site testing. Single-type fluorescent PCR testing requires two tubes, which is not only cumbersome and inefficient but also increases the risk of cross-contamination, failing to meet the needs of port quarantine, emergency clinical diagnosis, and large-scale epidemiological surveillance.

[0005] In summary, both Nipah virus type B and type M are highly pathogenic and have high mortality rates, and existing detection methods have many shortcomings. Therefore, developing a dual fluorescent PCR kit that can simultaneously detect both subtypes, is highly sensitive, time-efficient, and easy to operate, would enable early and rapid detection and typing, strengthen the monitoring of relevant occupational groups and susceptible populations, ensure timely detection and early intervention of infected individuals, reduce the risk of virus transmission, and has significant practical application value. Summary of the Invention

[0006] The purpose of this invention is to provide a dual fluorescent PCR kit that can simultaneously detect Nipah virus type B and type M, so as to solve the technical problems of existing detection methods that require single-type fluorescent PCR detection to be performed in two tubes, which is cumbersome, has low detection efficiency, and is prone to cross-contamination.

[0007] To achieve the above objectives, the present invention provides a dual fluorescent PCR kit for the simultaneous detection of Nipah virus type B and type M.

[0008] This kit contains a primer mixture that can simultaneously detect Nipah virus type B and type M, the primer mixture consisting of type B Nipah virus-specific primer pairs and type M Nipah virus-specific primer pairs.

[0009] Furthermore, the type B Nipah virus-specific primer pair consists of an upstream primer NiV-BF and a downstream primer NiV-BR, whose nucleotide sequences are shown in SEQ ID No. 03 and SEQ ID No. 04, respectively; the type M Nipah virus-specific primer pair consists of an upstream primer NiV-MF and a downstream primer NiV-MR, whose nucleotide sequences are shown in SEQ ID No. 01 and SEQ ID No. 02, respectively.

[0010] Furthermore, in the primer mixture, the concentrations of NiV-MF and NiV-MR are both 0.4 μM, and the concentrations of NiV-BF and NiV-BR are both 0.35 μM.

[0011] Furthermore, the kit also contains a nonspecific fluorescent dye selected from SYBR Green, Eva Green, Helix Green, and LC Green, preferably SYBR Green I.

[0012] Furthermore, the kit also includes an RT-PCR buffer containing the following components: M-MLV reverse transcriptase 4-6 U, Hot-Start Taq DNA polymerase 5-10 U, Tris-HCl 25-75 mM (pH 8.2-8.4), KCl 50-75 mM, MgCl2 2.5-3.75 mM, dATP 2.5-7.5 mM, dTTP 2.5-7.5 mM, dCTP 2.5-7.5 mM, dGTP 2.5-7.5 mM, RNase inhibitor 8-12 U, glycerol 2.5%-12.5%, and NaN 30.2%-1%.

[0013] Furthermore, the RT-PCR buffer contains the following components: 4U of M-MLV reverse transcriptase, 5U of Hot-StartTaq DNA polymerase, 50mM of Tris-HCl at pH 8.3, 50mM of KCl, 3mM of MgCl2, 4mM of dATP, 4mM of dTTP, 4mM of dCTP, 4mM of dGTP, 10U of RNase inhibitor, 10% glycerol, and 0.5% NaN.

[0014] Furthermore, the kit also includes a positive control and a negative control, wherein the positive control is prepared from a mixture of recombinant plasmids of the N gene of Nipah virus type B and Nipah virus type M, and the negative control is prepared from RNase / DNase-free TE buffer.

[0015] Furthermore, the reaction system of the kit is as follows: 12.5 μL RT-PCR buffer, 4 μL primer mixture, 5 μL RNA extract of the sample to be tested or positive standard, 3.5 μL enzyme-free water, and the total reaction volume is 25 μL.

[0016] Furthermore, the kit employs the following multiplex amplification program: reverse transcription at 50℃ for 10 minutes; pre-denaturation at 95℃ for 30 seconds; denaturation at 95℃ for 5 seconds, annealing at 58-62℃ for 25 seconds, for a total of 40-45 cycles; the melting curve program is 60℃→95℃, 0.5℃ / step. Preferably, the multiplex amplification program is: reverse transcription at 50℃ for 10 minutes; pre-denaturation at 95℃ for 30 seconds; denaturation at 95℃ for 5 seconds, annealing at 60℃ for 25 seconds, for a total of 45 cycles; the melting curve program is 60℃→95℃, 0.5℃ / step.

[0017] Furthermore, the kit distinguishes between Nipah virus type B and type M by the melting curve Tm value, where the Tm value of Nipah virus type M is 77.0℃ and the Tm value of Nipah virus type B is 81.5℃.

[0018] The beneficial effects of this invention are: 1. The kit described in this invention is easy to operate and has a short reaction time. Single-tube detection can complete the detection and typing of Nipah virus type B and type M within 60 minutes, without the need for tube splitting, which greatly improves detection efficiency and reduces the risk of cross-contamination.

[0019] 2. The kit described in this invention has high sensitivity, with a detection limit of 500 copies / mL for both type B and type M Nipah virus, enabling early detection of low concentrations of the virus and buying time for virus control.

[0020] 3. The kit described in this invention has high specificity. The primers are designed for the conserved regions of two Nipah virus subtypes and have no cross-reactivity with other related viruses such as Hendra virus, measles virus, mumps virus, and influenza virus, ensuring the accuracy of the test results.

[0021] 4. The kit described in this invention can directly distinguish between Nipah virus type B and type M by the melting curve Tm value, without the need for additional probes or sequencing steps, thus reducing detection costs. It is compatible with conventional real-time fluorescence quantitative PCR instruments, making it convenient for primary healthcare institutions and port quarantine departments to promote its use. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0023] Figure 1 This is an amplification curve of Nipah virus type M in Example 4 of the present invention.

[0024] Figure 2 This is a standard curve diagram of Nipah virus type M in Embodiment 4 of the present invention.

[0025] Figure 3 This is an amplification curve of Nipah virus type B in Example 4 of the present invention.

[0026] Figure 4 This is a standard curve diagram of Nipah virus type B in Embodiment 4 of the present invention.

[0027] Figure 5 This is a melting curve diagram of Nipah virus type B and type M in Embodiment 6 of the present invention. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should also be noted that, to make the embodiments more comprehensive, the following embodiments are the best and preferred embodiments, and those skilled in the art can use other alternative methods to implement some well-known technologies; moreover, the accompanying drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0029] Example 1: Primer Design and Screening The whole genome sequences of Nipah virus types B and M were downloaded from the GenBank database (https: / / www.ncbi.nlm.nih.gov / ), and highly conserved N gene sequences with subtype specificity were screened out. Multiple sequence alignments of the downloaded Nipah virus N gene sequences of types B and M were performed using Clustal software to determine the conserved regions and specific differential sites of the two subtypes. Multiple pairs of specific primers were designed for the conserved regions of the two subtypes using Oligo7 software. Through gradient PCR, melting curve analysis, and sensitivity testing, the optimal primer pairs with high amplification efficiency, strong specificity, and no cross-reactivity were selected.

[0030] The specific primer sequences and concentrations for Nipah virus type M obtained through screening are as follows: the nucleotide sequence of the upstream primer NiV-MF is shown in SEQ ID No. 01, with a concentration of 0.4 μM; the nucleotide sequence of the downstream primer NiV-MR is shown in SEQ ID No. 02, with a concentration of 0.4 μM. The specific primer sequences and concentrations for Nipah virus type B obtained through screening are as follows: the nucleotide sequence of the upstream primer NiV-BF is shown in SEQ ID No. 03, with a concentration of 0.35 μM; the nucleotide sequence of the downstream primer NiV-BR is shown in SEQ ID No. 04, with a concentration of 0.35 μM.

[0031] The sequences, lengths, and SEQ ID numbers of the primers described in this invention are summarized in Table 1.

[0032] Table 1 Primer Sequence Information Example 2: Optimization and preparation of RT-PCR buffer This invention optimizes the concentrations of various components of the RT-PCR buffer to determine the optimal formulation. The components of the RT-PCR buffer and their concentrations in this embodiment are shown in Table 2: Table 2 Concentrations of each component of RT-PCR buffer Example 3: Determination of RT-PCR Amplification Program This embodiment employs a rapid RT-PCR amplification program that combines the annealing and extension steps into one, shortening the detection time while ensuring amplification efficiency and specificity. The specific amplification program is shown in Table 3. Table 3 RT-PCR amplification program Example 4: Preparation of Standard Curve The Nipah virus type B and type M N gene recombinant plasmid standards were diluted to five concentration gradients using a 10-fold serial dilution method.7 copies / mL, 10 6 copies / mL, 10 5 copies / mL, 10 4 copies / mL, 10 3 (copies / mL), each gradient sample was repeated at least 5 times, and standard curve experiments were performed according to the RT-PCR system of Example 2 and the amplification program of Example 3. The slope of the standard curve and R were statistically analyzed. 2 Value and amplification efficiency.

[0033] The amplification curve of Nipah virus type M is as follows: Figure 1 As shown, the standard curve is as follows Figure 2 As shown; Nipah virus type B amplification curve as shown Figure 3 As shown, the standard curve is as follows Figure 4 As shown. Experimental results show that the standard curves R for the two subtypes of the virus are... 2 The values ​​were all ≥0.998, and the amplification efficiency was between 90% and 105%, indicating that the amplification stability and accuracy of this kit were good.

[0034] Example 5: Sensitivity Test The lowest concentration standard (10) from the standard curves of Nipah virus type B and M was used. 3 The samples were diluted to 500 copies / mL using a 2-fold serial dilution method. The samples at these two concentration gradients were tested 20 times each. The positive detection rate of each gradient sample was calculated, and the lowest gradient sample with a positive detection rate of 95% was taken as the detection limit of the kit.

[0035] The results are shown in Tables 4 and 5: Table 4. Results of Nipah virus type B sensitivity test Table 5. Results of Nipah virus type M sensitivity test Experimental results show that the detection limit of the kit for both Nipah virus type B and type M is 500 copies / mL, which is highly sensitive and can meet the needs of early detection of low concentrations of virus.

[0036] Example 6: Melting Curve Analysis This embodiment uses the melting curve method to genotype Nipah virus type B and type M. After specific RT-PCR amplification of the N gene of the two subtypes, melting curve analysis was performed. Due to significant differences in amplicon size and GC content between type B and type M Nipah viruses, the Tm values ​​of the melting curves for the two viruses show a significant shift. The type of virus infection can be accurately determined by using the Tm value of the melting curve.

[0037] Melting curves of Nipah virus types B and M are as follows: Figure 5 As shown in the figure. The experimental results show that the Tm value of Nipah virus type M is 77.0℃, and the Tm value of Nipah virus type B is 81.5℃. The difference in Tm values ​​between the two is significant and they can be clearly distinguished.

[0038] Example 7: Specificity Test Using the nucleic acids of Hendra virus, measles virus, mumps virus, influenza A virus, and influenza B virus as templates, RT-PCR detection was performed using the kit of this invention to observe whether specific amplification and specific melting peaks appeared.

[0039] Experimental results showed that no specific amplification or non-specific melting peaks were observed in any of the control viruses, indicating that the kit of the present invention has good specificity, no cross-reactivity, and can accurately distinguish Nipah virus from other related viruses.

[0040] This invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of this invention. To provide the public with a thorough understanding of this invention, specific details are described in detail in the following preferred embodiments; however, those skilled in the art will fully understand the invention even without these details. Furthermore, to avoid unnecessary misunderstanding of the essence of this invention, well-known methods, processes, procedures, components, and circuits are not described in detail.

[0041] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A dual fluorescent PCR kit for simultaneous detection of Nipah virus type B and type M, characterized in that, The kit contains a primer mixture capable of simultaneously detecting Nipah virus type B and type M, the primer mixture consisting of type B Nipah virus-specific primer pairs and type M Nipah virus-specific primer pairs.

2. The dual fluorescent PCR kit for simultaneous detection of Nipah virus type B and M as described in claim 1, characterized in that, The type B Nipah virus-specific primer pair consists of an upstream primer NiV-BF and a downstream primer NiV-BR, and their nucleotide sequences are shown in SEQ ID No. 03 and SEQ ID No. 04, respectively; the type M Nipah virus-specific primer pair consists of an upstream primer NiV-MF and a downstream primer NiV-MR, and their nucleotide sequences are shown in SEQ ID No. 01 and SEQ ID No. 02, respectively.

3. The dual fluorescent PCR kit for simultaneous detection of Nipah virus type B and type M according to claim 2, characterized in that, In the primer mixture, the concentrations of NiV-MF and NiV-MR were both 0.4 μM, and the concentrations of NiV-BF and NiV-BR were both 0.35 μM.

4. The dual fluorescent PCR kit for simultaneous detection of Nipah virus type B and M as described in claim 1, characterized in that, The kit also contains a nonspecific fluorescent dye selected from SYBR Green, Eva Green, Helix Green, and LC Green.

5. The dual fluorescent PCR kit for simultaneous detection of Nipah virus type B and type M according to claim 4, characterized in that, The fluorescent dye is SYBR Green I.

6. The dual fluorescent PCR kit for simultaneous detection of Nipah virus type B and M as described in claim 1, characterized in that, The kit also includes an RT-PCR buffer containing the following components: M-MLV reverse transcriptase 4-6 U, Hot-Start Taq DNA polymerase 5-10 U, Tris-HCl 25-75 mM (pH 8.2-8.4), KCl 50-75 mM, MgCl2 2.5-3.75 mM, dATP 2.5-7.5 mM, dTTP 2.5-7.5 mM, dCTP 2.5-7.5 mM, dGTP 2.5-7.5 mM, RNase inhibitor 8-12 U, glycerol 2.5%-12.5%, and NaN 30.2%-1%.

7. The dual fluorescent PCR kit for simultaneous detection of Nipah virus type B and M as described in claim 6, characterized in that, The RT-PCR buffer contains the following components: 4U M-MLV reverse transcriptase, 5U Hot-Start Taq DNA polymerase, 50mM Tris-HCl at pH 8.3, 50mM KCl, 3mM MgCl2, 4mM dATP, 4mM dTTP, 4mM dCTP, 4mM dGTP, 10U RNase inhibitor, 10% glycerol, and 0.5% NaN.

8. The dual fluorescent PCR kit for simultaneous detection of Nipah virus type B and M as described in claim 1, characterized in that, The kit also includes a positive control and a negative control. The positive control is prepared from a mixture of recombinant plasmids of the N gene of Nipah virus type B and Nipah virus type M, and the negative control is prepared from RNase / DNase-free TE buffer.

9. The dual fluorescent PCR kit for simultaneous detection of Nipah virus type B and M as described in claim 1, characterized in that, The reaction system of the kit is as follows: 12.5 μL RT-PCR buffer, 4 μL primer mixture, 5 μL RNA extract of the sample to be tested or positive standard, 3.5 μL enzyme-free water, and a total reaction volume of 25 μL.

10. The dual fluorescent PCR kit for simultaneous detection of Nipah virus type B and M according to claim 1, characterized in that, The kit uses the following multiplex amplification program: reverse transcription at 50℃ for 10 minutes; pre-denaturation at 95℃ for 30 seconds; denaturation at 95℃ for 5 seconds; annealing at 58-62℃ for 25 seconds, for a total of 40-45 cycles; the melting curve program is 60℃→95℃, 0.5℃ / step.

Images