CN111808998A - Adenovirus whole genome capture method, primer group and kit - Google Patents

Abstract

The invention relates to an adenovirus whole genome capture method, a primer group and a kit, which can conveniently and rapidly amplify adenovirus whole genome with a small amount of RNA, and can directly butt joint a second-generation sequencing library building reagent and a third-generation sequencing platform to obtain an adenovirus whole genome sequence.

Description

Adenovirus whole genome capture method, primer group and kit

Technical Field

The invention belongs to the field of virus detection, and particularly relates to an adenovirus whole genome capture method, a primer group and a kit.

Background

Human adenovirus (HAdV) pneumonia is one of the more serious types of community-acquired pneumonia of children, mostly occurs in children of 6 months to 5 years old, and some children have serious clinical manifestations, and pneumonia complications are many, and severe cases easily leave slow-moving airway and lung diseases, so that the Human adenovirus (HAdV) pneumonia is one of the important reasons for infant pneumonia death and disability at present.

HAdV belongs to the mammalian adenovirus genus, is a double-stranded DNA virus without envelope, and at least 90 genotypes have been found, which are divided into 7 sub-genera of a-G, and the tissue infectivity, pathogenicity, epidemic area, etc. of different types of HAdV are different. The HAdV related to respiratory tract infection mainly comprises subgenus B (type HAdV-3, 7, 11, 14, 16, 21, 50 and 55), subgenus C (type HAdV-1, 2, 5, 6 and 57) and subgenus E (type HAdV-4), adenovirus pneumonia accounts for 4-10% of community-acquired pneumonia, and severe pneumonia is common in types 3 and 7. The major prevalence in China is adenovirus type 3, 7, 11, 14, 55.

The method for detecting the adenovirus nucleic acid has higher sensitivity and specificity, mainly takes a fluorescence quantitative PCR method at present, and practice shows that the method has high detection specificity, low cost and convenient operation. However, the kit designs specific primers and probes according to known virus sequences, so that only known virus types can be identified, and unknown novel viruses cannot be identified; in addition, due to the variation of viral gene sequences, the amplification of primers and probes fails, the detection sensitivity is reduced, so the primers and the probes need to be replaced periodically and actual evaluation needs to be repeated, and the situation of adenovirus false negative identification by using a fluorescent quantitative PCR method often occurs.

In addition, a detection method based on metagenomic Next Generation Sequencing (abbreviated as "ngs" and also referred to as "metagenomics") can be used for identifying and diagnosing infection of other respiratory pathogens including adenovirus and realizing rapid detection of virus sequences. The metagenome-based next-generation sequencing technology (mNGS) does not depend on traditional microorganism culture, directly carries out high-throughput sequencing on nucleic acid in a clinical sample, can quickly and objectively detect various pathogenic microorganisms (including viruses, bacteria, fungi and parasites) in the clinical sample, and is particularly suitable for diagnosing critical and difficult infections. With the improvement of the mNGS technology platform and the increase of clinical research, the clinical application of the mNGS will become more and more extensive. In 2016, the United states FDA approved the use of the second generation sequencing technology for microorganism identification and drug-resistant virulence analysis; in 2019, pathogen metagenomics is written into diagnosis and treatment guidelines for adult hospital acquired pneumonia and ventilator associated pneumonia.

Sequencing refers to a method of determining the sequence of a gene. The genes of each species control the expression of a biological trait, and sequencing refers to the determination of the sequence of the genes. Second generation sequencing (NGS), also known as High-throughput sequencing, is a DNA sequencing technology developed based on PCR and gene chips. The sequencing technology has been developed to the third generation, which refers to single molecule sequencing technology. When DNA sequencing is carried out, PCR amplification is not needed, and each DNA molecule is independently sequenced. Third generation sequencing techniques are also called de novo sequencing techniques, i.e., single molecule real-time DNA sequencing. In recent years, new generation sequencing technologies (NGS) (also known as second generation sequencing) and Nanopore (Nanopore) (also known as third generation sequencing) have been increasingly applied to the diagnosis of unknown pathogens. The SARS-CoV-2 virus can obtain the complete sequence of virus genome in short time, and is also benefited by sequencing technology.

Generally, sequencing techniques have three roles in disease progression and outbreak: 1. identifying unknown pathogens, and determining the identity of new pathogens only if the whole genome of the pathogen is obtained; 2. the epidemic disease development stage can be used for directly detecting clinical samples, dynamically monitoring the mutation condition of the virus genome in real time and assisting the diagnosis of false negative samples; 3. and in the conventional detection stage, the method can be used for screening drug-resistant sites and can also be used for identifying daily respiratory tract infection, so that the detection accuracy of respiratory tract diseases is improved.

The current sequencing technology for identifying adenovirus comprises the following steps: the pathogen metagenome sequencing and the probe capture sequencing pathogen metagenome sequencing (mNGS) are the most common gene sequencing method aiming at the pathogen clinically at present, are culture-free and preference-free pathogen detection technologies based on the next-generation sequencing technology, and can complete the detection of various pathogens such as bacteria, fungi, viruses, parasites and the like at one time. The principle of probe capture is to artificially design a probe (in DNA or RNA form) that can be partially or fully complementary to the target segment. The sample and the probe are mixed, the probe captures the target segment, the segment without designed probe is eluted and discarded, and then the probe and the capture segment are separated by denaturation (usually pH is adjusted to be alkaline), and the captured segment can be used for the second generation sequencing library construction. However, the success rate of pathogen whole genome acquisition is influenced by various complex factors of application scenario difference, and meanwhile, the problems of time, manpower, high enterprise sequencing cost and the like of first-line personnel under task stress are also faced. Therefore, the pathogenic metagenome sequencing (mNGS) can be used for determining a negative sample, but the whole genome information is not easy to obtain, and the purposes of large-scale popularization and rapid diagnosis cannot be realized on the detection of the adenovirus due to the reasons of complex operation, relatively long detection time (required for 24-72 hours) and the like. As a technical supplement, the amplification-based genome capture method is a 'Culture-free' virus genome specific enrichment sequencing scheme, is simple and convenient to operate and low in cost, and is suitable for the current requirements of epidemic prevention and control, however, the existing probe capture sequencing method is complex in operation steps, mainly comprises 7 most of different technical operations, each part of operation needs more than 5 steps, more than 33 steps, and once operation is carried out, the operation in each part cannot be suspended and must be continuously completed in order to meet the detection requirements. This introduces a lot of uncontrollable and complicated operation to the actual operation.

Disclosure of Invention

The invention aims to provide a method for rapidly, conveniently and economically obtaining the whole genome of various adenovirus subtypes for disease control and scientific research personnel, and the method designs a plurality of pairs of shingled PCR amplification primers for the whole genome sequences of adenovirus type 3, 7, 11, 14 and 55 separated strains, and can conveniently and rapidly amplify the whole genome of various adenovirus subtypes by a small amount of RNA (ribonucleic acid) by adding a high-efficiency reverse transcription system and a high-fidelity Tag enzyme amplification system, thereby perfectly docking a second-generation sequencing library building reagent and a third-generation sequencing platform and rapidly and conveniently obtaining the whole genome sequence of 5 adenovirus subtypes.

In the first aspect of the invention, a sequence combination for adenovirus detection is provided, the sequence combination comprises 16 PCR amplification primers designed aiming at the complete sequence of an adenovirus genome, and the 16 amplification primer sequences sequentially comprise SEQ ID Nos. 1-16 or SEQ ID No:1-16 by one or more nucleotide substitution, deletion and/or insertion and has the same function with the corresponding SEQ ID No. 1-16; further preferably, the amplification primer design mode is shingled PCR.

Preferably, the sequence combination is designed according to adenovirus type 3, 7, 11, 14 and 55.

In a second aspect of the present invention, a kit is provided, which comprises a sequence combination for adenovirus detection, wherein the sequence combination comprises 16 PCR amplification primers designed for the complete sequence of adenovirus genome, and the 16 amplification primer sequences sequentially comprise SEQ ID nos. 1 to 16 or SEQ ID nos: 1-16 by one or more nucleotide substitution, deletion and/or insertion and has the same function with the corresponding SEQ ID No. 1-16; preferably, the amplification primer design mode is shingled PCR.

Preferably, the sequence combination is designed according to adenovirus type 3, 7, 11, 14 and 55.

Preferably, the kit further comprises an RT-PCR amplification system, and further preferably, a high efficiency reverse transcription system and a high fidelity Tag enzyme amplification system which are conventional in the art can be used in the present invention, and more preferably, the RT-PCR amplification system can be a commercial system, such as a SurSprit III ONE-STEP RT-PCR amplification system.

In a third aspect of the present invention, a sequencing fragment capture method for adenovirus detection is provided, which comprises the following specific steps:

(1) RNA extraction: extracting virus RNA from adenovirus in the specimen;

(2) primer design and PCR amplification: 16 amplification primers designed aiming at the conserved region of the adenovirus are used, and the sequence of the amplification primers comprises SEQ ID No.1-16 or SEQ ID No:1-16 and DNA molecules with the same function as the corresponding SEQ ID No.1-16 through substitution, deletion and/or insertion of one or more nucleotides, applying an RT-PCR amplification system, and taking the virus RNA extracted in the step (1) for rapid amplification;

(3) sequencing: after the amplification reaction is finished, adenovirus gene fragments with different sizes are obtained in the PCR reaction and are directly operated on a computer for sequencing.

Preferably, in the step (1), the strain specimen type includes nasopharyngeal swab, deep cough sputum, alveolar lavage fluid or lung tissue biopsy specimen.

Preferably, in the step (1), the final volume of RNA extraction is 20-50 ul.

Preferably, in the step (1), the viral RNA obtained after RNA extraction is a nucleic acid sample with a Ct value of less than 27.

Preferably, in the step (2), the amplification primer sequences are designed according to adenovirus types 3, 7, 11, 14 and 55.

Preferably, the RT-PCR amplification system in STEP (2) is a high efficiency reverse transcription system and a high fidelity Tag enzyme amplification system, further preferably, a high efficiency reverse transcription system and a high fidelity Tag enzyme amplification system which are conventional in the prior art can be used in the invention, and more preferably, the RT-PCR amplification system can be a commercial system, such as a SurSprit III ONE-STEP RT-PCR amplification system.

Preferably, in the step (2), the PCR amplification procedure includes: 25-35min at 45-55 deg.C, 2-3min at 94-96 deg.C, 15-30s at 53-56 deg.C, and 2min-3min at 70-75 deg.C for 35-40 reaction cycles; 10min at 72 ℃ and 10min at 4 ℃ Hold, more preferably 30min at 50 ℃, 2min at 94 ℃, 15s at 94 ℃, 30s at 55 ℃ and 2min at 72 ℃ for 30s, for a total of 40 reaction cycles; 72 ℃ for 10min,4 ℃ Hold.

In a fourth aspect of the invention, the application of the sequence combination in preparing a kit for detecting adenovirus is provided.

In the fifth aspect of the invention, a sequencing fragment capture method for adenovirus detection is provided, and sequencing fragments for adenovirus detection are obtained by using the kit.

According to the sixth aspect of the invention, the invention provides an adenovirus detection method, the sequence combination or the kit or the sequencing fragment capture method is used for obtaining the whole genome sequence of various subtypes of adenovirus, and the sequencing technology is used for detecting the adenovirus.

Specifically, the invention also relates to a sequencing fragment capturing method for adenovirus detection, which comprises the following specific steps:

(1) the specimen types are: nasopharyngeal swab, deep cough sputum, alveolar lavage fluid, lung tissue biopsy specimen, etc., the above specimens are used for whole genome capture after nucleic acid extraction;

(2) extracting adenovirus according to the corresponding requirements and steps in the virus RNA extraction kit, wherein the final volume is recommended to be 20-50 ul; the extracted virus RNA needs to be subjected to adenovirus qPCR detection, and the nucleic acid sample with the Ct value lower than 27 can be continuously subjected to the following operations for sequencing detection, and can also be stored at-70 ℃ for later use if the detection is not directly performed after the virus extraction, but repeated freeze thawing is avoided;

(3) 16 amplification primers designed aiming at adenovirus type 3, 7, 11, 14 and 55 conserved regions are used, the sequences of the primers are SEQ ID Nos. 1-16 in sequence, an RT-PCR amplification system is applied, a small amount of RNA is taken, the whole genome of the adenovirus is rapidly amplified, after the amplification reaction is finished, adenovirus gene segments with different sizes are obtained in the PCR reaction, and the adenovirus gene segments can be directly operated on a computer to perform a second-generation sequencing library building experiment and a third-generation sequencing platform.

The invention relates to a sequencing fragment capturing method, which belongs to the former stage of a sequencing process, does not judge a detection result, and belongs to a non-disease diagnosis method.

The capture method and the kit of the invention complete the reverse transcription process and the PCR process in one system, and have the advantages of convenient operation, short time and low requirement on sample quality. The reverse transcription and amplification process of the virus can be completed within 3 hours by only one RCR reaction, the library building reagent for the illumina needs only 10 hours from the nucleic acid to the extensive preparation, and the sequencing data needs only 5 hours from the extracted cassette nucleic acid to the obtained sequencing data by using a nanopore rapid library building method. At present, other kits by a capture method generally take more than 20 hours from nucleic acid to a library, and have complex operation and need more supporting equipment.

The invention can amplify samples with high virus content such as strains and the like, can also amplify samples with relatively low virus content such as throat swabs, sputum and the like, and basically can amplify the whole genome sequence as long as the CT value of the sample for the quantitative adenovirus qPCR is below 27.

The technical effects and advantages of the invention are summarized as follows:

(1) the operation is simple and convenient, the single-tube operation of nucleic acid transcription and amplification reduces complexity and reduces cross contamination risk;

(2) the amplification efficiency is high, and the genome coverage is uniform;

(3) the sample is directly detected without strain separation;

(4) the specific enrichment is carried out on the pathogenic sequence, the detection signal-to-noise ratio is improved, and the detection depth can be used for pathogen fine analysis of variation, drug resistance, propagation and evolution;

(5) the sequencing is economical, the required data amount is small, a single sample is less than 1M reads, and the method is suitable for all sequencing platforms such as illumina, Nanopore and the like.

Drawings

FIG. 1 shows the sequencing result of the ultrasensitive adenovirus whole genome capture method of the present invention.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

In each of the following examples, the equipment and materials were obtained from several companies as indicated below:

QIAamp Viral RNA Mini Kit purchased from QIAGEN;

the SurSprit III ONE-STEP RT-PCR amplification system was purchased from Invitrogen;

sequencer was purchased from Illumina, model Miseq;

sequencing systems were purchased from ThermoFisher, model Ion GeneStaudio S5.

Example 1 library construction sequencing based on ultrasensitive adenovirus whole genome Capture method

The invention specifically uses a multiplex PCR-based technology to carry out targeted amplification aiming at the gene sequence of the adenovirus, thereby improving the sequencing depth, effectively reducing the subsequent sequencing cost and shortening the research time. One key point in multiplex PCR technology is to design a reasonable multiplex PCR primer pair combination, ensure that there are no overlapping amplicons in the primer combination, and reduce the interactions between the primers. The method designs 16 primers, uses an Invitrogen SurSprit III ONE-STEP RT-PCR amplification system, takes a small amount of RNA, rapidly amplifies the whole genome of adenovirus, obtains adenovirus gene fragments with different sizes in PCR reaction after the amplification reaction is finished, and can be directly operated on a computer to perform a second-generation sequencing library building experiment and a third-generation sequencing platform.

The ultrasensitive adenovirus whole genome capture and library construction detection method specifically operates as follows:

first, materials and reagents:

isolating the strain; the specimen types are: nasopharyngeal swab, deep cough sputum, alveolar lavage fluid, lung tissue biopsy specimen, etc., the above specimens are used for whole genome capture after nucleic acid extraction.

Preparing a reaction system:

(1) taking out each reagent, mixing uniformly, putting into a centrifuge, and centrifuging for a short time.

(2) Each sample was prepared according to a 50. mu.l reaction per tube,

reaction system:

secondly, the method comprises the following steps:

(1) RNA extraction process: extracting adenovirus by adopting a Kit QIAamp Viral RNA Mini Kit according to the corresponding requirements and steps in the Viral RNA extraction Kit, wherein the Kit used in the operation flow is the QIAamp Viral RNA Mini Kit. The kit firstly cracks a sample under a high-denaturation condition, so that RNA enzyme is inactivated and the release of complete virus RNA is ensured, the optimal combination state of RNA and a silica gel matrix biomembrane is provided by adjusting the buffering environment, then the sample is loaded on a QIAamp centrifugal column, the RNA is combined on the biomembrane, and pollutants are effectively removed by two times of washing with different washing liquids. The final volume was 20-50 ul using RNase-free buffer. The extracted virus RNA is detected by qPCR, and the nucleic acid sample with the Ct value lower than 27 can be continuously subjected to the following operations for sequencing detection; if the virus is not directly detected after being extracted, the virus can be stored at-70 ℃ for later use, but repeated freezing and thawing are avoided.

(2) Primer design

The invention uses the shingled PCR amplification technology, belongs to a multiple PCR technology, is a means of target sequence enrichment, is used for capturing a plurality of sequence fragments, and the sequences are arranged like shingles, cover the whole length of a genome, and have the advantages of small data volume and high depth.

A plurality of pairs of amplification primers (shown in table 1) designed aiming at adenovirus type 3, 7, 11, 14 and 55 conserved regions are used, a SurSprit III ONE-STEP RT-PCR amplification system is applied, a small amount of RNA is taken, the whole genome of adenovirus is rapidly amplified, after the amplification reaction is finished, adenovirus gene segments with different sizes are obtained in the PCR reaction, and the adenovirus gene segments can be directly operated on a computer to perform a second-generation sequencing library building experiment and a third-generation sequencing platform.

The PCR amplification procedure was as follows:

setting sequencing cycle parameters:

TABLE 1 primer sequence information

(3) Sequencing process

After the amplification reaction is finished, adenovirus gene segments with different sizes are obtained in one tube of PCR reaction, and 2 tubes of amplification products are concentrated in one tube after the experiment is finished. Purifying the amplified product, wherein the purified sample with the qubit quantitative of more than 10ng/ul can be directly subjected to subsequent library building experiments, and then the subsequent library building experiments are carried out on a sequencing platform, such as a Miseq sequencer or a second generation or third generation sequencing platform such as an Ion GeneStudios5 sequencing system.

The sequencing result is analyzed by respiratory virus whole genome analysis software, the sequencing result covers 100% of the genome, and the sequencing depth of all positions reaches more than 100X. Exemplary sequencing results are shown in figure 1.

Example 2 ultrasensitive adenovirus whole genome Capture method of the invention compared to other methods

The capture method of the invention can amplify samples with high virus content such as strains and the like, can also amplify samples with relatively low virus content such as throat swabs, sputum and the like, and can amplify the whole genome sequence as long as the quantitative CT value of the sample for adenovirus qPCR is below 27.

The ultrasensitive adenovirus whole genome capture method of the present invention was compared with other methods available, as shown in Table 2 (in the table "-" represents no detection). The results show that the whole genome sequence cannot be well obtained by the existing methods such as a direct RNA library construction method and a direct reverse transcription library construction method, the operation time of the capture method after library construction and the direct RNA library construction method is longer than that of the invention, and the detection cannot be realized in the aspects of sequencing coverage degree and the like although the operation time of the reverse transcription library construction method is short. Therefore, the ultrasensitive library construction detection method and the kit amplification and use capture method can be used for detecting the whole genome, have great advantages in both library construction time and capture efficiency, and can accurately and efficiently obtain the whole genome of the virus in large-scale population detection.

TABLE 2 comparison of the ultrasensitive adenovirus whole genome capture method of the present invention with other methods available

Although the present invention has been described with respect to the preferred embodiments, it is not intended to be limited to the embodiments disclosed, and many modifications and variations are possible to those skilled in the art without departing from the spirit of the invention.

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Claims (11)

1. The sequence combination for detecting the adenovirus is characterized by comprising 16 imbricated PCR amplification primers designed aiming at the complete sequence of an adenovirus genome, wherein the 16 amplification primer sequences sequentially comprise SEQ ID No.1-16 or SEQ ID No:1-16 through one or several nucleotide substitution, deletion and/or insertion and has the same function with the corresponding SEQ ID No. 1-16.

2. The combination of sequences for adenovirus detection according to claim 1, wherein said combination of sequences is designed according to adenovirus type 3, 7, 11, 14, 55.

3. A kit comprises a sequence combination for adenovirus detection, and is characterized in that the sequence combination comprises 16 imbricated PCR amplification primers designed aiming at the complete sequence of an adenovirus genome, and the 16 amplification primer sequences sequentially comprise SEQ ID No.1-16 or SEQ ID No:1-16 by one or more nucleotide substitution, deletion and/or insertion and has the same function with the corresponding SEQ ID No. 1-16.

4. The kit of claim 3, wherein the combination of sequences is designed according to adenovirus type 3, 7, 11, 14, or 55.

5. A sequencing fragment capture method for adenovirus detection is characterized by comprising the following specific steps:

(1) RNA extraction: extracting virus RNA from adenovirus in the specimen;

(2) primer design and PCR amplification: 16 amplification primers designed aiming at the conserved region of the adenovirus are used, and the sequence of the amplification primers comprises SEQ ID No.1-16 or SEQ ID No:1-16 and DNA molecules with the same function as the corresponding SEQ ID No.1-16 through substitution, deletion and/or insertion of one or more nucleotides, applying an RT-PCR amplification system, and taking the virus RNA extracted in the step (1) for amplification;

(3) sequencing: after the amplification reaction is finished, adenovirus gene segments with different sizes are obtained in the PCR reaction and are directly sequenced.

6. The capturing method as claimed in claim 5, wherein in the step (1), the strain specimen type includes nasopharyngeal swab, deep sputum, alveolar lavage or lung tissue biopsy specimen.

7. The method of claim 5 or 6, wherein the final volume of RNA extraction in step (1) is 20-50 ul.

8. The capturing method according to claim 5 or 6, wherein in the step (1), the viral RNA obtained after RNA extraction is a nucleic acid sample with a Ct value lower than 27.

9. The method of claim 5, wherein in step (2), the amplification primer sequences are designed according to adenovirus type 3, 7, 11, 14, and 55.

10. Use of a combination of sequences according to claim 1 or 2 for the preparation of a kit for the detection of adenovirus.

11. A sequencing fragment capture method for adenovirus detection, characterized in that the sequencing fragment for adenovirus detection is obtained by using the kit of claim 3 or 4.

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