WO2018192193A1

WO2018192193A1 - Lamp primer combination for detecting six respiratory viruses and use thereof

Info

Publication number: WO2018192193A1
Application number: PCT/CN2017/105455
Authority: WO
Inventors: 郝荣章; 王瑞丽; 宋宏彬; 孔文; 李杨; 赵荣涛; 孙明璇; 贾雷立; 邱少富; 郭旭东
Original assignee: 中国人民解放军疾病预防控制所
Priority date: 2017-04-18
Filing date: 2017-10-10
Publication date: 2018-10-25
Also published as: CN106884012B; CN106884012A

Abstract

The present invention provides a LAMP primer combination for detecting six respiratory viruses and use thereof. The primer combination consists of DNA molecules as shown in sequence 1 to sequence 36, and is used for detecting influenza A H1N1 virus, influenza A H3N2 virus, H5N1 avian influenza virus, H7N9 avian influenza virus, influenza B virus and human adenovirus.

Description

LAMP primer combination for detecting six respiratory viruses and its application

Technical field

The invention belongs to the field of biotechnology, and particularly relates to a LAMP primer combination for detecting six respiratory viruses and an application thereof.

Background technique

Respiratory infections are classified into upper respiratory tract infections and lower respiratory tract infections. About 90% of upper respiratory tract infections are caused by viruses, and bacterial infections are often secondary to viral infections. The disease can occur in all seasons and at any age, and spreads through droplets, mists, or contaminated utensils containing viruses. Whether it is a lower respiratory tract infection or an upper respiratory tract infection, most of it is caused by a virus called a viral respiratory infection.

The influenza virus belongs to the Orthomyxoviridae family and is classified into influenza A virus (such as influenza A H1N1 influenza virus, influenza A H3N2 influenza virus, H5N1 avian influenza virus, H7N9 avian influenza virus, etc.), influenza B virus and influenza C virus III. Kind. Influenza virus is a serious threat to human health and can infect a wide range of hosts. The annual seasonal epidemic causes about 600 million infections worldwide and causes 250,000-500,000 deaths. The main cause of infection and death is influenza A. virus. In the past 100 years, the flu virus has caused at least four pandemics worldwide, all of which are caused by animal-borne influenza viruses that infect people across the host barrier. In recent years, H5N1 avian influenza virus and H7N9 avian influenza virus have been reported in the world. For example, H5N1 avian influenza outbreak in Hong Kong in 1997 directly infected people and caused death. In 2013, H7N9 avian influenza broke out in Shanghai and Anhui. More than 300 people were infected and more than 50 died. These events prove that the flu virus not only causes huge economic losses, but also directly infects humans across the host barrier, posing a serious threat to human public health security.

Adenovirus is a non-encapsulated particle with a diameter of 70-90 nm, which consists of 252 shells arranged in a squamous plane. The linear double-stranded DNA molecule is in the capsid. Human adenovirus (HAdV) is a member of the mammalian adenovirus family of the adenoviridae family. Human adenovirus is one of the most important pathogens causing acute respiratory infections in infants and young children. The main epidemic strains causing acute respiratory infections are HAdV-3 (human adenovirus type 3) and HAdV-7 (human adenovirus type 7), which can also cause diseases in other systems, and the clinical manifestations of the disease are related to other respiratory viruses. The virus is similar, but more with gastrointestinal symptoms. Adenovirus infections have occurred in many countries and regions around the world. In China, serious outbreaks of adenovirus infection occurred in the last century.

The prevalence of respiratory viruses can pose major security problems for the entire human society. The rapid detection of respiratory viruses is of great significance for the prevention and control of major infectious diseases and bioterrorism. The rapid detection technology for the development of multiple pathogens of respiratory viruses is the key to timely discovering the source of infection and implementing timely and effective isolation and control of infectious sources. In recent years, molecular diagnostic methods (such as polymerase chain reaction) have been used for the detection of respiratory viruses, which can accurately identify target sequences, but it requires components of thermal cycling systems, which are often dependent on large instruments and are expensive. Not conducive to rapid detection.

Loop-mediated isothermal amplification (LAMP) is a chain of denaturation and automatic cycling of nucleic acids under isothermal conditions using a Bst DNA polymerase with strand displacement activity and waterfall nucleic acid amplification. Replace the nucleic acid amplification reaction. LAMP designs 4 special features for 6 regions of the target sequence

Claims

A heterologous primer that continuously replicates amplified DNA at a constant temperature using a DNA polymerase having a strand displacement function. In order to improve the reaction efficiency, two loop primers may be added to the reaction system to bind to the stem-loop structure, start strand displacement synthesis, and cyclic replication. LAMP has the advantages of simplicity, rapidity, sensitivity and specificity, and is especially suitable for the application of LAMP kits in the grassroots. In LAMP technology, primers are a key factor in determining the sensitivity and specificity of the test results.

Invention disclosure

The technical problem to be solved by the present invention is how to detect influenza A H1N1 virus, influenza A H3N2 virus, H5N1 avian influenza virus, H7N9 avian influenza virus, influenza B virus and human adenovirus.

In order to solve the above technical problems, the present invention first provides a primer combination which may be as follows (a1) or (a2) or (a3):

(a1) consisting of primer set I, primer set II, primer set III, primer set IV, primer set V, and primer set VI;

(a2) any two, any three, any of the primer set I, the primer set II, the primer set III, the primer set IV, the primer set V, and the primer set VI Four or any five components;

(a3) the primer set I, the primer set II, the primer set III, the primer set IV, the primer set V or the primer set VI.

The primer set I may be composed of H1N1-F3, H1N1-B3, H1N1-FIP, H1N1-BIP, H1N1-LF and H1N1-LB; the H1N1-F3 may be as follows (b1) or (b2): (b1) a single-stranded DNA molecule as shown in SEQ ID NO: 1 of the Sequence Listing; (b2) a DNA molecule having one or more nucleotide substitutions and/or deletions and/or additions and having the same function as Sequence 1; H1N1-B3 may be a single-stranded DNA molecule as shown in SEQ ID NO: 2 of the sequence listing (b3) or (b4): (b3); (b4) substitution and/or deletion of sequence 2 by one or several nucleotides And/or a DNA molecule which has the same function as that of the sequence 2; the H1N1-FIP may be a single-stranded DNA molecule represented by the sequence 3 of the sequence table (b5) or (b6): (b5); (b6) Sequence 3 is subjected to one or several nucleotide substitutions and/or deletions and/or additions of DNA molecules having the same function as Sequence 3; said H1N1-BIP may be as follows (b7) or (b8): (b7 a single-stranded DNA molecule as shown in SEQ ID NO: 4 of the Sequence Listing; (b8) a DNA molecule having a sequence 4 having a substitution and/or deletion and/or addition of one or several nucleotides and having the same function as that of SEQ ID NO:4; H1N1-LF can be as follows (b9) or (b10): (b9) sequence a single-stranded DNA molecule as shown in SEQ ID NO: 5; (b10) a DNA molecule which has undergone substitution and/or deletion and/or addition of sequence 5 with one or several nucleotides and which has the same function as that of SEQ ID NO: 5; -LB may be a single-stranded DNA molecule as shown in SEQ ID NO: 6 of the sequence listing (b11) or (b12): (b11); (b12) a substitution and/or deletion of sequence 6 by one or several nucleotides / or a DNA molecule added and having the same function as that of SEQ ID NO: 6.

The primer set II may be composed of H3N2-F3, H3N2-B3, H3N2-FIP, H3N2-BIP, H3N2-LF and H3N2-LB; the H3N2-F3 may be as follows (c1) or (c2): (c1) a single-stranded DNA molecule as shown in SEQ ID NO: 7; (c2) a DNA molecule having sequence 7 substituted and/or deleted and/or added and having the same function as SEQ ID NO: 7; H3N2-B3 may be a single-stranded DNA molecule as shown in SEQ ID NO: 8 of the sequence listing (c3) or (c4): (c3); (c4) substitution and/or deletion of sequence 8 by one or several nucleotides And/or a DNA molecule having the same function as that of the sequence 8; the H3N2-FIP may be a single-stranded DNA molecule represented by the sequence 9 of the sequence listing (c5) or (c6): (c5); (c6) Pass sequence 9 through one or several nucleotides