CN116355896A

CN116355896A - Primer probe for detection, primer probe group and application thereof

Info

Publication number: CN116355896A
Application number: CN202111615153.3A
Authority: CN
Inventors: 赵雨航; 胡莞尔; 詹浩淼; 唐放
Original assignee: Maccura Biotechnology Co ltd
Current assignee: Maccura Biotechnology Co ltd
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2023-06-30

Abstract

The invention relates to a primer probe for PCR detection, a primer probe group and application thereof. The primer probe and the primer probe group can simultaneously analyze two dimensions of a fluorescence channel and a melting temperature in a single-tube reaction, namely, different targets can be detected through the melting temperature characteristic of the same fluorescence channel; or different fluorescent channels are used, so that target species detection of the product of the number of the fluorescent channels and the melting temperature characteristic can be realized. The method for carrying out multiplex PCR detection by utilizing the primer probe has the advantages of low fluorescence background, adjustable melting temperature, good inclusion, low cost, single tube reaction, simple and convenient operation, difficult pollution, high sensitivity and wide application range.

Description

Primer probe for detection, primer probe group and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a primer probe for PCR detection, a primer probe group and application thereof.

Background

Polymerase Chain Reaction (PCR) is a molecular biological technique that performs enzymatic replication of DNA without using living organisms. PCR is commonly used in medical and biological research laboratories to undertake a variety of tasks such as diagnosis of infectious diseases, gene cloning, phenotypic identification of experimental animals, transcriptome studies, detection of genetic diseases, identification of gene fingerprints, paternity test, and the like. PCR is considered by molecular biologists as the method of choice for nucleic acid detection due to its incomparable replication and precision capabilities. In the late 90 s of the last century, the real-time fluorescent quantitative PCR (Real Time Quantitative PCR, qPCR) technology and related products proposed by the American ABI company have developed PCR into a highly sensitive, highly specific and precisely quantitative nucleic acid sequence analysis technology.

At present, a more primer probe design mode is mainly a TaqMan hydrolysis probe method on a qPCR platform, and the working principle is mainly that an oligonucleotide probe which can be specifically combined with a template and is respectively marked with a fluorescent group (donor) and a quenching group (acceptor) at two ends is utilized, and meanwhile, a specific PCR primer is respectively designed at the upstream and the downstream of the probe. Before the PCR reaction starts, due to the Fluorescence Resonance Energy Transfer (FRET) principle, a fluorescent signal emitted by a fluorescent group at one end of the TaqMan probe is absorbed by a quenching group at the other end, so that the fluorescent signal is not detected by an instrument; and after PCR amplification starts, the TaqMan probe is specifically combined with the template, when the DNA polymerase (Taq enzyme) extends to the site of the probe combined with the template, the 5-3' exonuclease activity of the TaqMan probe cuts off the TaqMan probe, so that a fluorescent group marked on the probe is far away from a quenching group and no FRET structure is formed any more, and therefore, a signal emitted by the fluorescent group can be detected by an instrument. However, in the same reaction system, in order to realize PCR detection of multiple targets, a plurality of TaqMan hydrolysis probes for marking fluorescent groups with different wavelengths are required to be arranged, and at most, 4-6 different targets can be detected only according to the number of fluorescent channels of a detection instrument.

To achieve ultra-multiplexed PCR detection, the prior art generally combines PCR detection techniques with other methods, such as PCR with hybridization chips, to perform multiplex target detection with different amplification product lengths. The method for detecting the kit by combining multiple RT-PCR (reverse transcription-polymerase chain reaction) with gene chips of common pathogens of respiratory tract disclosed in document CN107090519A utilizes the principle of nucleic acid molecular hybridization, single-stranded probes for each target are arranged and fixed on the gene chips according to a specific sequence to form a probe array, and multiple PCR products to be detected are hybridized with the probe array, so that the target amplification products are hybridized with the probes on the chips and emit fluorescent signals. Although the method can detect 20 respiratory pathogens simultaneously, the preparation process of the gene chip is complex, expensive, unfavorable for clinical popularization and development, complicated in process, and easy to cause pollution to cause false positive of results due to the fact that a plurality of steps of uncovering and cleaning are needed.

As disclosed in CN103074450B, a kit for synchronously detecting thirty diarrhea pathogenic bacteria and a detection method thereof utilize a mode of combining PCR amplification and capillary electrophoresis, take out PCR amplification products and perform capillary electrophoresis analysis, and compare the obtained spectrum with a standard spectrum to determine the kind of diarrhea pathogenic bacteria. In the method, besides the PCR amplification detection equipment, capillary electrophoresis equipment is also required to be used for product analysis, so that the detection cost is greatly increased, and the method is not beneficial to clinical popularization and use.

Therefore, a method capable of carrying out multiplex molecular detection is urgently needed in clinic, and various clinically common targets such as bacteria, viruses, gene mutation and the like can be detected rapidly, simply and conveniently at low cost, so that clinical units including primary hospitals can rapidly carry out detection of common targets such as pathogenic microorganism infection detection and the like, other detection and diagnosis means are assisted, diagnostic evidence and medication schemes are provided for clinic more rapidly, mental burden and economic burden of patients are reduced, and the aim of accurate medical treatment is achieved.

Disclosure of Invention

The invention provides a primer probe and a primer probe group for PCR detection, which can effectively avoid false positive caused by primer dimer.

To this end, the first aspect of the present invention provides a primer probe for PCR detection comprising a forward primer (F), a probe (P) and a reverse primer (R);

the forward primer (F) comprises a target sequence binding region 1, wherein the target sequence binding region 1 is a sequence capable of specifically pairing and binding with a target sequence;

The probe (P) is a sequence which is not in pairing with any target sequence and comprises a probe signal detection region (H); the probe (P) is modified with a detection group;

the reverse primer (R) comprises a primer signal detection region (H) and a target sequence binding region 2, wherein the target sequence binding region 2 is a sequence capable of specifically pairing and binding with a target sequence, the primer signal detection region (H) is a sequence which does not pairing and binding with any target sequence, and the primer signal detection region (H) has a part or all of the same sequence as the probe signal detection region (H) of the probe (P);

the forward primer (F) and the reverse primer (R) are respectively combined with a target in a specific way to generate a pre-amplification product, the pre-amplification product contains a single-chain pre-amplification product with a reverse complementary sequence (h ') of a primer signal detection area (h), the reverse complementary sequence (h') of the single-chain pre-amplification product is combined with a probe (P) in a specific way and extends to form a secondary amplification double-chain product, and the formation of the secondary amplification double-chain product causes the probe to generate a detectable signal change;

wherein at least one of the forward primer (F) and the reverse primer (R) consists of two separate sequences.

In some embodiments of the invention, the forward primer (F) consists of two separate sequences and the reverse primer (R) consists of one separate sequence;

The first sequence of the forward primer (F) sequentially comprises an amplification product capturing region (B) and a target sequence binding region 1 from the 5 'end to the 3' end, the second sequence of the forward primer (F) comprises the amplification product capturing region (B), and the amplification product capturing region (B) is a sequence which is not in pairing combination with any target sequence;

the reverse primer (R) comprises a primer signal detection region (h) and a target sequence binding region 2 from the 5 'end to the 3' end in sequence.

In the present invention, when the forward primer (F) is composed of two separate sequences, competition of the forward primer (F) with the pre-amplification product for the probe can be reduced.

In some embodiments of the invention, the first sequence of the forward primer (F) has a 0 to 20 base separation between the amplification product capture region (B) and the target sequence binding region 1;

in other specific embodiments of the present invention, the reverse primer (R) has a 0 to 20 base interval between the primer signal detection region (h) and the target sequence binding region 2.

In some preferred embodiments of the present invention, the second sequence of the forward primer (F) comprises, in order from the 5 'end to the 3' end, a probe anchoring region (a) and an amplification product capturing region (B);

the probe (P) is a sequence which is not in pairing connection with any target sequence and comprises a primer anchor region (A') and a probe signal detection region (H); the primer anchor region (A') is a sequence complementary to the probe anchor region (A).

The term "the primer anchor region (A') is complementary to the probe anchor region (A)" means that both can be complementary in the forward direction or in the reverse direction, so long as the complementary pairing is possible.

In some embodiments of the invention, the first sequence of the forward primer (F) is 20 to 80 bases in length, wherein the Tm value of the target sequence binding region 1 is 40 to 80℃and the GC content is 20 to 80%; the length of the amplified product capturing zone (B) is 5-35 bases, the Tm value is 40-80 ℃, and the GC content is 20-80%.

In some embodiments of the invention, the second sequence of the forward primer (F) is 20 to 80 bases in length, wherein the probe anchoring region (A) is 6 to 35 bases in length, the Tm value is 40 to 80 ℃, and the GC content is 30 to 80%; the length of the amplified product capturing zone (B) is 5-35 bases, the Tm value is 40-80 ℃, and the GC content is 20-80%.

In some embodiments of the invention, the reverse primer (R) is 20 to 80 bases in length, wherein the Tm value of the target sequence binding region 2 is 40℃to 80℃and the GC content is 20% to 80%; the length of the primer signal detection region (h) is 5-65 bases, the Tm value is 30-80 ℃, and the GC content is 20-80%.

In other embodiments of the invention, the probe (P) is 20 to 100 bases in length, wherein the primer anchor region (A') is 6 to 35 bases in length, the Tm is 40 to 80 ℃, and the GC content is 30 to 80%; the length of the probe signal detection area (H) is 5-65 bases, the Tm value is 30-80 ℃, and the GC content is 20-80%.

In some embodiments of the invention, the forward primer (F) and the reverse primer (R) are each composed of two separate sequences;

the first sequence of the reverse primer (R) sequentially comprises a reverse amplification product capturing region (C) and a target sequence binding region 2 from the 5 'end to the 3' end, the second sequence of the reverse primer (R) sequentially comprises a primer signal detecting region (h) and a reverse amplification product capturing region (C) from the 5 'end to the 3' end, and the reverse amplification product capturing region (C) is a sequence which is not in pairing combination with any target sequence.

in other embodiments of the present invention, the first sequence of the reverse primer (R) has a 0 to 20 base interval between the reverse amplification product capture region (C) and the target sequence binding region 2.

In some embodiments of the invention, the first sequence of the reverse primer (R) is 20 to 80 bases in length, wherein the Tm value of the target sequence binding region 2 is 40 to 80℃and the GC content is 20 to 80%; the length of the reverse amplification product capturing zone (C) is 5-35 bases, the Tm value is 40-80 ℃, and the GC content is 30-80%.

In other embodiments of the invention, the second sequence of the reverse primer (R) is 20 to 80 bases in length and has a GC content of 20 to 80%; wherein the length of the primer signal detection region (h) is 5-65 bases, the Tm value is 30-80 ℃, and the GC content is 20-80%; the length of the reverse amplification product capturing zone (C) is 5-35 bases, the Tm value is 40-80 ℃, and the GC content is 30-80%.

In some embodiments of the invention, the probe (P) is 40 to 100 bases in length, wherein the primer anchor region (A') is 6 to 35 bases in length, the Tm value is 40 to 80 ℃, and the GC content is 40 to 80%; the length of the probe signal detection area (H) is 5-65 bases, the Tm value is 40-80 ℃, and the GC content is 40-80%.

In some embodiments of the invention, the detection moiety comprises a first detection moiety and a second detection moiety, and the first detection moiety and the second detection moiety produce a change in signal by a change in distance; preferably, the first detection group and the second detection group are separated by 5-25 bases; further preferably, the first detection moiety is a fluorescent reporter moiety and the second detection moiety is a quencher moiety or other modifying moiety capable of generating a signal change from the first detection moiety via fluorescence resonance energy transfer.

In the present invention, the first and second detection groups are positioned on the probe such that the reverse complement (h') of the single-stranded pre-amplification product specifically binds to the probe (P) and extends to form a double-stranded secondary amplification product, the formation of which causes a detectable signal change to the probe. For example, at least 1 detection group is modified at the 5' end of the probe signal detection zone (H) of the probe, or at least 1 detection group is modified within the probe signal detection zone (H) of the probe.

In the present invention, the primer anchor region (A ') may be designed at the 5' end of the probe (P) or at the 3' end of the probe (P). In some embodiments of the invention, the probe (P) comprises, in order from the 5' end to the 3' end, a primer-anchor region (a ') and a probe signal detection region (H); preferably, the detection group on the probe (P) is modified between the primer anchor region (A') and the probe signal detection region (H).

In other embodiments of the present invention, the probe (P) comprises a probe signal detection region (H) and a primer-anchor region (a ') in order from the 5' end to the 3' end; preferably, the detection group on the probe (P) is modified at the 5' end of the probe signal detection zone (H).

In some embodiments of the invention, the 3' end of the probe (P) contains a blocking region.

In the present invention, the blocking region (blocking region); i.e., a moiety that serves to prevent extension of a nucleic acid strand by a DNA polymerase, thereby preventing strand extension during, for example, PCR. The blocking region may be a 3'OH modified with 3' -Spacer C3, 3 '-phoshate, 3' -ddC, 3 '-introduced End, etc., so that the 3' OH is blocked, thereby preventing the extension reaction. The blocking region may be a polymerase blocking group, or a group that blocks further extended functional properties of the polymer. The blocking group can be any chemical group capable of attaching to a nucleotide that will allow the 5' end of the modified nucleotide to attach to the 3' end of another nucleotide in the DNA strand but will not allow the nucleotide to attach to the 3' hydroxyl group of the modified nucleotide. Suitably, the absence of an OH group at the 3' position will prevent further extension by the polymerase activity. In some embodiments, the blocking group is selected from acetyl, CH3, glycyl, leucyl, and alanyl groups. In other embodiments, the blocking group may be in the form of a di-or tripeptide.

In some embodiments of the invention, the Tm value of the primer signaling detection region (h) is less than both the Tm value of the target sequence binding region 1 in the forward primer (F) and the Tm value of the target sequence binding region 2 in the reverse primer (R).

In other embodiments of the invention, the Tm value of the primer anchor region (a') is greater than the Tm value of the primer signal detection region (h).

In the present invention, the generation of primer dimer (dimer) during amplification can be effectively avoided by limiting the Tm value of the primer signal detection region (h) and the Tm value of the primer anchor region (A') to the above-described ranges.

Notably, are: even if a dimer is generated during the amplification process, the signals of the dimer can be distinguished by a melting curve, and if the signals are collected at a higher dissolution temperature, the signals of the dimer cannot be collected. The specific method is as follows: the signal change generated by the double-stranded product formed by the specific combination of the reverse complementary sequence (h ') of the single-stranded pre-amplification product and the probe (P) is different from the signal change generated by the double-stranded product formed by the specific combination of the reverse complementary sequence (h') of the single-stranded pre-amplification product and the probe (P) and the extension, wherein the signal change is different in signal strength and/or melting temperature.

In some preferred embodiments of the present invention, the primer probe includes:

a forward primer (F) consisting of two separate sequences; wherein the first sequence of the forward primer (F) sequentially comprises an amplification product capturing region (B) and a target sequence binding region 1 from the 5 'end to the 3' end, wherein the target sequence binding region 1 is a sequence capable of being specifically matched and bound with a target sequence, the amplification product capturing region (B) is a free design sequence which is not matched and bound with any target sequence, and a base interval of 0-20 can be arranged between the amplification product capturing region (B) and the target sequence binding region 1; the second sequence of the forward primer (F) sequentially comprises a probe anchoring region (A) and an amplified product capturing region (B) from the 5 'end to the 3' end;

a probe (P) which is a free design sequence that does not bind with any target sequence in a pairing manner, and comprises a primer anchor region (A '), wherein the other parts except the primer anchor region (A') are probe signal detection regions (H); the sequence of the primer anchor region (A') is complementary to the probe anchor region (A) sequence of the second sequence of the forward primer (F); modifying a second detection group at a base position 5-25 bases from the first detection group, both the first detection group and the second detection group being on the same side of the primer anchor region (A'), wherein the first detection group may be a fluorescent reporter group and the second detection group may be a quencher group or other modification group capable of generating a signal change with the first detection group by Fluorescence Resonance Energy Transfer (FRET);

A reverse primer (R) consisting of a single sequence comprising, in order from the 5 'end to the 3' end, a primer-signal detection region (H) and a target-sequence binding region 2, wherein the target-sequence binding region 2 is a sequence capable of specifically pairing-binding with a target sequence, and the primer-signal detection region (H) is a free design sequence that does not pair-bind with any target sequence, but has a sequence that is partially or wholly identical to the probe-signal detection region (H) of the probe (P); the primer signal detection region (h) and the target sequence binding region 2 may have a base interval of 0 to 20.

In the invention, the principle of PCR detection of a sample to be detected by using the primer probe is as follows:

in PCR amplification, a forward primer (F) and a reverse primer (R) in a primer probe are respectively combined with a target sequence in a specific way and extend to generate a first double-stranded amplification product, wherein the 5' end to the 3' end of one first single-stranded amplification product (S1) is sequentially provided with a primer signal detection region (h), a target sequence and a reverse complementary sequence (B ') of an amplification product capture region; the amplification product capturing region (B) in the second sequence of the forward primer (F) can be reversely complemented and extended with the reverse complement sequence (B ') of the amplification product capturing region at the 3' end of one of the first single-stranded amplification products (S1) to obtain a second double-stranded amplification product, wherein the reverse complement sequence (h ') of the probe anchoring region (A), the pre-amplification product capturing region (B), the target sequence and the primer signal detection region (h) is sequentially arranged from the 5' end to the 3' end of one of the second single-stranded amplification products (D1);

After the second double-stranded amplification product is generated, the probe signal detection region (H) of the probe (P) is complementarily paired with the reverse complement sequence (H ') of the primer signal detection region at the 3' end of one of the second single-stranded amplification products (D1), the primer anchoring region (A ') of the probe (P) is complementarily paired with the probe anchoring region (A) at the 5' end of one of the second single-stranded amplification products (D1), the 3 'end of one of the second single-stranded amplification products (D1) can continue to extend to the 5' end of the probe (P) to obtain a part or all of the reverse complement sequence of the probe (P), and amplification by taking one of the second single-stranded amplification products (D1) as a primer and the probe (P) as a template is completed to obtain an amplified double-stranded product formed by the probe (P). At this time, the probe (P) is changed from a single-stranded state to a double-stranded state, and the distance between the first detection group and the second detection group is changed to generate a signal which can be detected by an instrument;

after the PCR amplification is finished, melting curve analysis is carried out, in the process of temperature change, amplified double-strand products formed by the probe (P) are melted at a certain temperature, and at the moment, the probe (P) with the first detection group and the second detection group is changed from double-strand to single-strand state, so that the distance between the detection groups is changed, a signal is generated, and the signal can be detected by an instrument. The signal detected by the instrument presents a melting curve along with the temperature change, and when the Tm value of the peak position of the melting curve is within the range of the characteristic Tm value of a specific detection channel of a target object in a sample to be detected, the sample to be detected is indicated to contain the target object; the specific detection channel is a fluorescent channel corresponding to the first detection group on the probe (P), and the characteristic Tm value is the melting temperature of a double-strand product of secondary amplification formed by the probe (P).

In other preferred embodiments of the present invention, the primer probe includes:

a probe (P) which is a free design sequence that does not bind with any target sequence in a pairing manner, and comprises a primer anchor region (A '), wherein the other parts except the primer anchor region (A') are probe signal detection regions (H); the sequence of the primer anchor region (A') is complementary to the probe anchor region (A) sequence of the second sequence of the forward primer (F); modifying a first detection group at a base position of 1-95 bases from the primer anchor region (A '), and modifying a second detection group at a base position 5-25 bases from the first detection group, both the first detection group and the second detection group being on the same side of the primer anchor region (A'), wherein the first detection group can be a fluorescent reporter group and the second detection group can be a quencher group or other modification group capable of generating a signal change with the first detection group by Fluorescence Resonance Energy Transfer (FRET);

A reverse primer (R) consisting of two separate sequences; wherein the first sequence of the reverse primer (R) comprises a reverse amplification product capturing region (C) and a target sequence binding region 2 in sequence from the 5 'end to the 3' end, wherein the target sequence binding region 2 is a sequence capable of specifically pairing and binding with a target sequence, the reverse amplification product capturing region (C) is a free design sequence which is not pairing and binding with any target sequence, and a base interval of 0-20 can be arranged between the reverse amplification product capturing region (C) and the target sequence binding region 2; the second sequence of the reverse primer (R) sequentially comprises a primer signal detection region (h) and a reverse amplification product capture region (C) from the 5 'end to the 3' end, wherein the primer signal detection region (h) is a free design sequence which is not in pairing combination with any target sequence.

in PCR amplification, a forward primer (F) and a reverse primer (R) in a primer probe are respectively combined with a target sequence in a specific way and extend to generate a first double-stranded amplification product, wherein one first single-stranded amplification product (S1) is a forward amplification product capturing region (B), a target sequence and a reverse complementary sequence (C ') of the reverse amplification product capturing region from a 5' end to a 3' end in sequence, and the other first single-stranded amplification product (S1 ') is a reverse amplification product capturing region (C), a target sequence and a reverse complementary sequence (B ') of the forward amplification product capturing region in sequence from the 5' end to the 3' end;

When the PCR amplification is continued, the first double-stranded amplification product is used as a template, and the forward amplification product capturing region (B) in the second sequence of the forward primer (F) can be reversely complementary and extended with the reverse complementary sequence (B ') of the forward amplification product capturing region of the other first single-stranded amplification product (S1'); the reverse amplification product capture region (C) in the second sequence of the reverse primer (R) may be reverse-complementary to and extend from the reverse complement (C') of the reverse amplification product capture region of one of the first single-stranded amplification products (S1) to yield a second double-stranded amplification product. The 5 'end to the 3' end of one second single-stranded amplification product (D1) is sequentially provided with a probe anchoring region (A), a forward amplification product capturing region (B), a target sequence, a reverse complementary sequence (C ') of the reverse amplification product capturing region and a reverse complementary sequence (h') of a primer signal detecting region;

after the second double-stranded amplification product is generated, the probe signal detection region (H) of the probe (P) is complementarily paired with the reverse complement sequence (H ') of the primer signal detection region at the 3' end of one of the second single-stranded amplification products (D1), the primer anchoring region (A ') of the probe (P) is complementarily paired with the probe anchoring region (A) at the 5' end of one of the second single-stranded amplification products (D1), the 3 'end of the second single-stranded amplification product (D1) can continue to extend to the 5' end of the probe (P) to obtain part or all of the reverse complement sequence of the probe (P), and amplification by taking one of the second single-stranded amplification products (D1) as a primer and the probe (P) as a template is completed to obtain an amplified double-stranded product formed by the probe (P); at this time, the probe (P) is changed from a single-stranded state to a double-stranded state, and the distance between the first detection group and the second detection group is changed to generate a signal which can be detected by an instrument;

Notably, are: the sequence structures of the forward primer and the reaction primer in the present invention may be interchanged. For example, the forward primer (F) comprises a primer signal detection region (h) and a target sequence binding region, and the reverse primer comprises a target sequence binding region; alternatively, the forward primer (F) comprises a primer signal detection region (h) and a target sequence binding region, and the reverse primer (R) comprises a probe anchor region (a) and a target sequence binding region.

In a second aspect, the invention provides a kit for PCR detection comprising a primer probe for PCR detection according to the first aspect of the invention; preferably, further comprising an amplification reagent; further preferably, the amplification reagents comprise DNA polymerase and dNTPs; still further preferably, when the nucleic acid template is RNA, the amplification reagent further comprises reverse transcriptase.

In the present invention, the amplification reagents may further include reagents for promoting PCR reaction, such as KCl, mgCl ₂ Tris-HCl, dithiothreitol (DTT), (NH) ₄ ) ₂ SO ₄ Etc.

In a third aspect, the present invention provides a primer probe set for PCR detection, comprising at least 1 probe (P), at least 2 forward primers (F) and at least 2 reverse primers (R);

each of the different forward primers (F) independently comprises a target sequence binding region 1 that specifically binds to a different target sequence pair;

the probe (P) is a sequence which is not in pairing with any target sequence and comprises a probe signal detection region (H); the sequence of the probe signal detection region (H) of different probes (P) is different, and the modified detection groups are different;

each of the different reverse primers (R) independently comprises a primer signal detection region (h) and a target sequence binding region 2; wherein the target sequence binding region 2 in the different reverse primers (R) is a sequence which is specifically matched and bound with different target sequences, and the primer signal detection regions (H) in the different reverse primers (R) are all a section of a sequence which is not matched and bound with any target sequences but is partially or completely the same as the probe signal detection region (H) of the probe (P);

The forward primer (F) and the reverse primer (R) are respectively combined with a target in a specific way to generate a pre-amplification product, the pre-amplification product contains a single-stranded pre-amplification product with a reverse complementary sequence (h ') of a primer signal detection area (h), the reverse complementary sequence (h') in the single-stranded pre-amplification product is combined with a probe (P) in a specific way and extends to form a secondary amplification double-strand product, and the formation of the secondary amplification double-strand product causes the signal change of the probe;

the single-stranded preamplification products formed by the different forward primers (F) and the reverse primers (R) are different, and the Tm values of the secondary amplification double-stranded products formed by the different single-stranded preamplification products are different;

wherein at least one forward primer (F) and/or the corresponding reverse primer (R) consists of two separate sequences;

preferably, the sequences of the primer signal detection regions (h) of different said reverse primers (R) are different from each other.

In the present invention, "the sequences of the primer-signal detection regions (h) of the different reverse primers (R) are different from each other" means that the bases and/or the lengths of the sequences of the primer-signal detection regions (h) of the different reverse primers (R) are different so that different single-stranded pre-amplification products containing the reverse complement sequences (h') of the different primer-signal detection regions (h) bind to the probe and extend to form different double-stranded products for secondary amplification, and the melting temperature curves of the different double-stranded products for secondary amplification can be separated from each other.

Since the melting temperature of the double-stranded product formed with the probe (P) is detected during the melting curve analysis, the melting temperature of the double-stranded product formed with the probe (P) can be adjusted by adjusting the base and length of the sequence of the primer signal detection region (H) of the reverse primer (R) to obtain the reverse complementary sequence (H ') of the primer signal detection region (S1) of the single-stranded pre-amplified product partially reverse-complementary or fully reverse-complementary to the probe signal detection region (H) of the probe (P), for example, adjusting the length of the primer signal detection region (H) or the base of the sequence, or adjusting the position of the reverse complementary sequence (H') of the primer signal detection region and the probe signal detection region (H) of the probe (P) to increase or decrease the melting temperature of the double-stranded product formed with the probe (P).

In the invention, the primer probe group is provided with a plurality of groups of forward primers (F) and reverse primers (R) aiming at different target sequences, and when the primer probe group is used for multiplex PCR detection, different target sequences can be identified through fluorescent channels or through melting temperature; when different target sequences are identified by fluorescence channel, different probes (P) can be used for different target sequences, and when different target sequences are identified by melting temperature, different target sequences can share the same probe (P).

In some embodiments of the invention, the at least 1 forward primer (F) consists of two separate sequences and the reverse primer (R) corresponding to the forward primer (F) consists of one separate sequence;

the first sequence of the at least 1 forward primer (F) comprises an amplification product capturing region (B) and a target sequence binding region 1 from the 5 'end to the 3' end in sequence, the second sequence of the at least 1 forward primer (F) comprises an amplification product capturing region (B), and the amplification product capturing region (B) is a sequence which is not in pairing combination with any target sequence; the sequence of the target sequence binding region 1 in the different forward primers (F) is different; the amplification product capture zones (B) in different forward primers (F) may be the same or different;

the reverse primer (R) corresponding to the forward primer (F) sequentially comprises a primer signal detection region (h) and a target sequence binding region 2 from the 5 'end to the 3' end, and the sequences of the target sequence binding regions 2 of different reverse primers (R) are different.

In some embodiments of the invention, the first sequence of the different forward primer (F) has a 0 to 20 base interval between the amplification product capture region (B) and the target sequence binding region 1.

In other embodiments of the present invention, the reverse primer (R) is different in that there is a 0 to 20 base interval between the primer signal detection region (h) and the target sequence binding region 2.

In some preferred embodiments of the present invention, the second sequence of the at least 1 forward primer (F) comprises, in order from the 5 'end to the 3' end, a probe anchor region (a) and an amplification product capture region (B);

In some preferred embodiments of the invention, the primer anchor region (A') and/or the probe signal detection region (H) of different said probes (P) differ in sequence and the modified detection groups differ.

In the present invention, the probe anchor regions (A) of different forward primers may be the same or different. When the probe-anchoring regions (A) of the different forward primers are different, the sequences and/or lengths of the probe-anchoring regions A of the different forward primers and the primer-signal detection regions of the different reverse primers are such that the melting temperature curves of the double-stranded product formed by the (later) and the probe can be separated from each other. In some embodiments of the invention, the first sequence of the different forward primer (F) is 20 to 80 bases in length, wherein the Tm value of the target sequence binding region 1 is 40 to 80℃and the GC content is 20 to 80%; the length of the amplified product capturing zone (B) is 5-35 bases, the Tm value is 40-80 ℃, and the GC content is 20-80%.

In some embodiments of the invention, the second sequence of the different forward primer (F) is 20 to 80 bases in length, wherein the probe anchoring region (A) is 6 to 35 bases in length, the Tm value is 40 to 80 ℃, and the GC content is 30 to 80%; the length of the amplified product capturing zone (B) is 5-35 bases, the Tm value is 40-80 ℃, and the GC content is 20-80%.

In some embodiments of the invention, the different reverse primers (R) are 20 to 80 bases in length, wherein the Tm value of the target sequence binding region 2 is 40 to 80℃and the GC content is 20 to 80%; the length of the primer signal detection region (h) is 5-65 bases, the Tm value is 30-80 ℃, and the GC content is 20-80%.

In some embodiments of the invention, the at least 1 forward primer (F) and its corresponding reverse primer (R) are each composed of two separate sequences;

The first sequence of the at least 1 forward primer (F) comprises an amplification product capturing region (B) and a target sequence binding region 1 from the 5 'end to the 3' end in sequence, the second sequence of the at least 1 forward primer (F) comprises an amplification product capturing region (B), and the amplification product capturing region (B) is a sequence which is not in pairing combination with any target sequence; the sequence of the target sequence binding region 1 in the different forward primers (F) is different;

the first sequence of the reverse primer (R) corresponding to the at least 1 forward primer sequentially comprises a reverse amplification product capturing region (C) and a target sequence binding region 2 from the 5 'end to the 3' end, the second sequence of the reverse primer (R) corresponding to the at least 1 forward primer sequentially comprises a primer signal detecting region (h) and a reverse amplification product capturing region (C) from the 5 'end to the 3' end, and the reverse amplification product capturing region (C) is a sequence which is not in pairing combination with any target sequence; the sequence of the target sequence binding region 2 of the different reverse primer (R) is different.

In other embodiments of the invention, the reverse amplification product capture region (C) in the first sequence of the different reverse primer (R) is separated from the target sequence binding region 2 by 0 to 20 bases.

In some preferred embodiments of the invention, the second sequence of the different forward primer (F) comprises, in order from the 5 'end to the 3' end, a probe anchor region (a) and an amplification product capture region (B);

In the present invention, the probe anchor regions (A) of different forward primers may be the same or different. When the probe anchor regions (A) of the different forward primers are different, the sequences and/or lengths of the probe anchor regions A of the different forward primers and the primer signal detection regions of the different reverse primers are such that the different single-stranded pre-amplification products contain the reverse complementary sequences (h') of the different probe anchor regions (A) and the primer signal detection regions (h), the different single-stranded pre-amplification products bind to the probe and form different double-stranded products for secondary amplification after extension, and the melting temperature curves of the different double-stranded products for secondary amplification can be separated from each other.

Since the melting temperature of the double-stranded product of the secondary amplification with the probe (P) is detected at the time of melting curve analysis, the melting temperature of the double-stranded product of the secondary amplification with the probe (P) can be adjusted by adjusting the base and length of the sequence of the probe anchor region (A) of the forward primer (F) and/or the primer signal detection region (h) of the reverse primer (R). For example, the melting temperature of the double-stranded product of the secondary amplification formed with the probe (P) is increased or decreased by adjusting the base of the length or sequence of the probe anchor region, or the base of the length or sequence of the primer signal detection region (H), or adjusting the position where the probe anchor region is complementary to the primer anchor region (A ') of the probe (P), or adjusting the position where the reverse complementary sequence (H') of the primer signal detection region is reverse complementary to the probe signal detection region (H) of the probe (P).

In some embodiments of the invention, the first sequence of the different forward primer (F) is 20 to 80 bases in length, wherein the Tm value of the target sequence binding region 1 is 40 to 80℃and the GC content is 20 to 80%; the length of the amplified product capturing zone (B) is 5-35 bases, the Tm value is 40-80 ℃, and the GC content is 20-80%.

In other embodiments of the invention, the different probes (P) are 20 to 100 bases in length, wherein the primer anchor region (A') is 6 to 35 bases in length, the Tm is 40 to 80 ℃, and the GC content is 30 to 80%; the length of the probe signal detection area (H) is 5-65 bases, the Tm value is 30-80 ℃, and the GC content is 20-80%.

In other embodiments of the invention, the Tm value of the primer signaling detection region (h) is less than both the Tm value of the target sequence binding region 1 in the forward primer (F) and the Tm value of the target sequence binding region 2 in the reverse primer (R).

In some embodiments of the invention, the Tm value of the primer anchor region (a') is greater than the Tm value of the primer signal detection region (h).

In a fourth aspect, the present invention provides a kit for PCR detection comprising a primer probe set for PCR detection according to the third aspect of the present invention; preferably, further comprising an amplification reagent; further preferably, the amplification reagents comprise DNA polymerase and dNTPs; still further preferably, when the nucleic acid template is RNA, the amplification reagent further comprises reverse transcriptase.

In a fifth aspect, the present invention provides a method for PCR detection of a sample to be detected using a kit according to the second or fourth aspect of the present invention, comprising the steps of:

s1, mixing a forward primer (F), a reverse primer (R) and a probe (P) to form primer probe premix;

s2, mixing the primer probe premix with an amplification reagent and a sample to be detected to obtain a reaction system;

s3, carrying out PCR amplification on the reaction system;

s4, analyzing whether the target object exists.

In some embodiments of the present invention, the step S4 includes the steps of:

s4-1, obtaining signal changes generated by PCR amplification in the step S3, and judging whether a target object exists in the sample to be detected according to the signal changes; and/or

S4-2, performing melting curve analysis on the PCR amplified product in the step S3, and judging whether the sample to be detected contains a target object.

In some embodiments of the invention, the concentration of the first sequence of the forward primer (F) in the reaction system obtained in step S2 is 30nM to 1000nM, the concentration of the reverse primer (R) is 30nM to 500nM, and the concentration of the second sequence of the forward primer (F) is 30nM to 1000nM; the concentration of the probe (P) is 150nM-1200nM; or alternatively

The concentration of the first sequence of the forward primer (F) in the reaction system obtained in the step S2 is 30nM to 1000nM, the concentration of the first sequence of the reverse primer (R) is 30nM to 500nM, and the concentration of the second sequence of the forward primer (F) is 30nM to 1000nM; the concentration of the second sequence of the reverse primer (R) is 30nM to 1000nM; the concentration of the probe (P) is 150nM-1200nM.

In some embodiments of the invention, in step S3, when the nucleic acid template is DNA, the PCR amplification procedure is as follows:

pre-denaturation at 90-96 ℃ for 2-15 minutes; denaturation at 90-95 ℃ for 10-60 seconds, annealing at 50-75 ℃ and extension for 30-90 seconds, and 35-50 cycles; denaturation at 90-95 deg.c for 10-600 sec, annealing at 30-55 deg.c and extension for 10-600 sec, and 1-20 cycles.

In some embodiments of the invention, in step S3, when the nucleic acid template is DNA, the PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94℃for 10 seconds, annealing at 56℃for 30 seconds and extension for 45 cycles in total; denaturation at 94℃for 150 sec, annealing at 40℃and extension for 300 sec, 1 cycle.

In some embodiments of the invention, in step S3, when the nucleic acid template is RNA, the PCR amplification procedure is as follows:

Reverse transcription is carried out for 5-15 minutes at 50-60 ℃; pre-denaturation at 90-96 ℃ for 2-15 minutes; denaturation at 90-95 ℃ for 10-60 seconds, annealing at 50-75 ℃ and extension for 30-90 seconds, and 35-50 cycles; denaturation at 90-95 deg.c for 10-600 sec, annealing at 30-55 deg.c and extension for 10-600 sec, and 1-20 cycles. .

In some embodiments of the invention, in step S3, when the nucleic acid template is RNA, the PCR amplification procedure is as follows: reverse transcription at 55℃for 15 min; pre-denaturation at 95 ℃ for 3 min; denaturation at 94℃for 10 seconds, annealing at 56℃for 30 seconds and extension for 45 cycles in total; denaturation at 94℃for 150 sec, annealing at 40℃and extension for 300 sec, 1 cycle.

In a sixth aspect, the present invention provides a method for PCR detection of a sample to be detected using a kit according to the second or fourth aspect of the present invention, comprising the steps of:

specifically binding the forward primer (F) and the reverse primer (R) with a target respectively, and generating a pre-amplification product after amplification, wherein the pre-amplification product contains a reverse complementary sequence (h') of a primer signal detection region (h);

specifically binding the reverse complementary sequence (h') of the primer signal detection region (h) with the probe (P) and extending to form a secondary amplification double-chain product;

The formation of the double-chain product of the secondary amplification causes the probe to generate detectable signal change, and whether a target object exists in the sample to be detected is judged according to the signal change; and/or

And (3) carrying out melting curve analysis on the double-chain product of the secondary amplification and judging whether the sample to be detected contains a target object or not.

In some embodiments of the invention, the specific operation of the melting curve analysis is: gradually heating the temperature from 35-45 ℃ to 85-95 ℃, collecting fluorescent signals in real time in the heating process, and obtaining a melting curve after the heating is finished; preferably, the rate of temperature rise is 0.03 to 0.08 ℃/s.

In some embodiments of the invention, the melting curve analysis is performed by: gradually heating the temperature from 40 ℃ to 85-87 ℃, collecting fluorescent signals in real time in the heating process, and obtaining a melting curve after the heating is finished; the rate of temperature rise is 0.05 ℃/s.

In some embodiments of the present invention, when the Tm value of the peak position of the obtained melting curve is within the range of the Tm value of the target in the sample to be detected, the Tm value of the target in the sample to be detected is within the range of the Tm value of the characteristic detection channel, the Tm value of the peak position of the melting curve indicates that the sample to be detected contains the target; preferably, the specific detection channel is a fluorescent channel corresponding to the first detection group on the probe (P), and the characteristic Tm value is a melting temperature of a double-strand product of the secondary amplification formed by the probe (P).

In some embodiments of the invention, in the absence of the target to be detected, the primer anchor region (a') of the probe (P) is complementary to the probe anchor region (a) in the second sequence of the forward primer (F), the other part of the probe (P) is in a single-stranded state, and the first detection group and the second detection group are closer in distance due to the coiled state of the flexibility of the molecule, so that the fluorescence resonance energy transfer efficiency is higher; when the target to be detected exists, the forward primer (F) and the reverse primer (R) are respectively combined with the target sequence in a specific way and extend to generate a first double-stranded amplification product, wherein the 5' end to the 3' end of one first single-stranded amplification product (S1) is sequentially provided with a primer signal detection region (h), a target sequence and a reverse complementary sequence (B ') of an amplification product capture region; the amplification product capturing region (B) of the second sequence of the forward primer (F) can be reversely complemented and extended with the reverse complement sequence (B ') of the amplification product capturing region of the 3' -end of one of the first single-stranded amplification products (S1) to obtain a second double-stranded amplification product, wherein the reverse complement sequence (h ') of the probe anchoring region (A), the amplification product capturing region (B), the target sequence and the primer signal detection region is sequentially arranged from the 5' -end to the 3' -end of one of the second single-stranded amplification products (D1); the probe signal detection region (H) of the probe (P) is complementarily paired with the reverse complement sequence (H ') of the primer signal detection region at the 3' end of one second single-stranded amplification product (D1), the primer anchoring region (A ') of the probe (P) is complementarily paired with the probe anchoring region (A) at the 5' end of one second single-stranded amplification product (D1), the 3 'end of one second single-stranded amplification product (D1) can extend to the 5' end of the probe (P) continuously, so that partial or all reverse complement sequences of the probe (P) are obtained, namely amplification by taking one second single-stranded amplification product (D1) as a primer and taking the probe (P) as a template is completed, so that an amplified double-stranded product formed by the probe (P) is obtained, at the moment, the distance between the first detection group and the second detection group is prolonged due to the change of single strand to double strand, the fluorescence resonance energy transfer efficiency is reduced, and the fluorescence signal can be detected by an instrument; after the PCR is finished, when the melting curve analysis is carried out, the amplified double-stranded product formed by the probe (P) and the probe (P) is melted at a certain temperature, so that the probe (P) with the first detection group and the second detection group is changed into a single-stranded state, at the moment, the distance between the first detection group and the second detection group is shortened, the fluorescence resonance energy transfer efficiency is increased, and the fluorescence signal is changed again, so that the melting curve analysis can be used for the melting curve analysis. When the detected Tm value of the peak position of the melting curve is in the range of the characteristic Tm value of the target object in the sample to be detected in a specific detection channel, indicating that the sample to be detected contains the target object; the specific detection channel is a fluorescent channel corresponding to the first detection group on the probe (P), and the characteristic Tm value is the melting temperature of a double-strand product of secondary amplification formed by the probe (P).

In other embodiments of the invention, the primer anchor region (A') of the probe (P) is complementary to the probe anchor region (A) in the second sequence of the forward primer (F) in the absence of the target to be detected, and the other part of the probe (P) is in a single-stranded state, and the first detection group and the second detection group are closer in distance due to the coiled state of the flexibility of the molecule, so that the fluorescence resonance energy transfer efficiency is higher; when the target to be detected exists, the forward primer (F) and the reverse primer (R) are respectively combined with the target sequence in a specific way and extend to generate a first double-stranded amplification product, the second sequence of the forward primer (F) and the second sequence of the reverse primer (R) are further amplified with the first amplified double-stranded product to generate a second double-stranded amplification product, wherein the 5 '-end to the 3' -end of one second single-stranded amplification product (D1) is sequentially provided with a probe anchoring region (A), a forward amplification product capturing region (B), a target sequence, a reverse complementary sequence (C ') of the reverse amplification product capturing region and a reverse complementary sequence (h') of a primer signal detecting region; the probe signal detection region (H) of the probe (P) is complementarily paired with the reverse complement sequence (H ') of the primer signal detection region at the 3' -end of one of the second single-stranded amplification products (D1); the primer anchor region (A ') of the probe is complementarily paired with the probe anchor region (A) at the 5' end of one second single-stranded amplification product (D1), wherein the 3 'end of one second single-stranded amplification product (D1) can continue to extend to the 5' end of the probe (P) to obtain a part or all of reverse complementary sequences of the probe (P), namely, the amplification by taking one second single-stranded amplification product (D1) as a primer and the probe (P) as a template is completed, so that an amplified double-stranded product formed by the probe (P) is obtained, at the moment, the distance between the first detection group and the second detection group is prolonged due to the change from single strand to double strand, the fluorescence resonance energy transfer efficiency is reduced, a fluorescence signal is changed, and the fluorescence signal can be detected by an instrument; after the PCR is finished, when the melting curve analysis is carried out, the amplified double-stranded product formed by the probe (P) and the probe (P) is melted at a certain temperature, so that the probe (P) with the first detection group and the second detection group is changed into a single-stranded state, at the moment, the distance between the first detection group and the second detection group is shortened, the fluorescence resonance energy transfer efficiency is increased, and the fluorescence signal is changed again, so that the melting curve analysis can be used for the melting curve analysis. When the detected Tm value of the peak position of the melting curve is in the range of the characteristic Tm value of the target object in the sample to be detected in a specific detection channel, indicating that the sample to be detected contains the target object; the specific detection channel is a fluorescent channel corresponding to the first detection group on the probe (P), and the characteristic Tm value is the melting temperature of a double-strand product of secondary amplification formed by the probe (P).

In some embodiments of the invention, the sample to be tested is selected from any one of a serum sample, a plasma sample, a whole blood sample, a sputum sample, a swab sample, an lavage sample, a fresh tissue sample, and a formalin-fixed paraffin embedded tissue.

In some embodiments of the invention, the apparatus and reagent consumables used in the PCR amplification may be Shanghai Marble medical technology Co., ltd.

In other embodiments of the present invention, the detection results of the present invention may be analyzed by using SLAN real-time fluorescence quantitative PCR analysis software from Shanghai Marble medical science and technology Co.

In the present invention, the target may be at least one selected from Adenovirus (ADV), influenza B Virus (IBV), mycoplasma pneumoniae (Mycoplasma pneumoniae, MP), 2019 novel coronavirus (SARS-CoV-2), respiratory syncytial virus (Respiratory Syncytial Virus, RSV), influenza a virus (Influenza A virus, IAV), non-small cell lung cancer, and the like.

The beneficial effects of the invention are as follows:

(1) Multiplex detection: the primer probe and the primer probe group can simultaneously analyze two dimensions of a fluorescence channel and a melting temperature in a single-tube reaction, namely, different targets can be detected through the melting temperature characteristic of the same fluorescence channel; or different fluorescent channels are used, so that target species detection of the product of the number of the fluorescent channels and the melting temperature characteristic can be realized;

(2) The fluorescent background is low: each fluorescent channel can be distinguished by using only 1 probe and different melting temperatures of amplified products, so that the fluorescent background in the PCR reaction is greatly reduced, and the reaction sensitivity is improved;

(3) The melting temperature can be adjusted: the detection method using the primer probe of the invention is to distinguish by utilizing different melting temperatures of the secondary amplification products, so that the melting temperature of the secondary amplification double-chain products formed by the probe (P) is increased or reduced by adjusting the length or sequence of the primer signal detection area (H) or adjusting the reverse complementary position of the primer signal detection area (H) and the probe signal detection area (H) of the probe (P);

(4) The inclusion is good: the primer probe has two parts of reverse complementary pairing with the target sequence, namely a forward primer (F) and a reverse primer (R), and compared with the Taqman hydrolysis probe method, the three parts of the primer probe need to be in reverse complementary pairing with a template, and when more high variation regions exist in the target sequence, such as virus or bacterial genome, the inclusion of the primer probe is better, and the design difficulty is lower;

(5) The cost is low: the method provided by the invention has the advantages that the detection is finished in a single-tube reaction only through fluorescent quantitative PCR, the assistance of an additional downstream instrument is not needed, the split reaction of a chip or a plurality of reaction tubes is not needed, the cost of instruments and consumables is greatly reduced, meanwhile, the number of fluorescent probes in a reagent is reduced, the reagent cost is greatly reduced, and the large-scale popularization is possible;

(6) Single tube reaction: the consumption of the sample is low, the method is particularly suitable for detecting rare samples, the concentration of the sample added into the detection reaction can be greatly improved, the detection sensitivity is improved, and meanwhile, the single-tube reaction provides possibility for the subsequent extraction-free one-tube detection;

(7) The operation is simple and convenient: only a fluorescent quantitative PCR instrument is used for detection, no subsequent steps such as capillary electrophoresis or nucleic acid hybridization are needed, meanwhile, the preparation is simple, and no operation is needed after the machine is started;

(8) Is not easy to cause pollution: the method is completely closed after sample addition is completed, and subsequent detection is carried out without uncovering, so that the possibility of pollution to experimental environment is greatly reduced;

(9) The sensitivity is high: the method of the invention has very low requirement on the length of the target sequence, and when the target type is short-fragment nucleic acid, such as free nucleic acid, the shorter length of the target sequence has higher sensitivity in detection;

(10) The application range is wide: the method of the invention is applicable to nucleic acid detection of various sample types, including serum samples, plasma samples, whole blood samples, sputum samples, swab samples, lavage samples, fresh tissue samples, formalin-fixed paraffin embedded tissue (FFPE).

Drawings

The invention will be further described with reference to the accompanying drawings.

FIG. 1 is a melting curve of the single PCR reaction of example 1 positive for RSV (71.64 ℃) in the CY5 channel.

FIG. 2 is a melting curve of the RSV (82.32 ℃) positivity of the multiplex PCR reaction of example 2 in the VIC channel.

Detailed Description

In order that the invention may be more readily understood, the invention will be further described in detail with reference to the following examples, which are given by way of illustration only and are not limiting in scope of application. The starting materials or components used in the present invention may be prepared by commercial or conventional methods unless specifically indicated.

Example 1: single PCR detection of Respiratory Syncytial Virus (RSV)

(1) The base sequences of primer probes for detecting Respiratory Syncytial Virus (RSV) by multiplex PCR are shown in table 1.

Table 1: base sequence of primer probe

Among the above primer probes:

the first sequence (SEQ ID NO: 3) of the forward primer F1-1 is a specific primer designed for a target sequence of respiratory syncytial virus, the full length of the first sequence of F1-1 is 44bp, the 1 st to 29 th bases at the 3' end are a target sequence binding region 1, the 1 st to 12 th bases at the 5' end are an amplified product capturing region (B), and the 3' end of the first sequence (SEQ ID NO: 4) of the forward primer F1-1 is the same as the 1 st to 12 th base sequence;

The reverse primer R1-1 (SEQ ID NO: 2) is a specific primer designed for a target sequence of respiratory syncytial virus, the total length of R1-1 is 35bp, the 1 st to 24 th bases at the 3' end of the reverse primer are a target sequence binding region 2, the 1 st to 8 th bases at the 5' end of the reverse primer are a primer signal detection region (h), and the sequence of the 1 st to 8 th bases at the 3' end of the probe P1-1 (SEQ ID NO: 1) is the same;

the second sequence (SEQ ID NO: 4) of the forward primer F1-1 has a total length of 25bp, the 1 st to 12 th bases at the 3 'end thereof are amplification product capturing regions (B), the 1 st to 10 th bases at the 5' end thereof are probe anchoring regions (A), and the 1 st to 10 th base sequences at the 5 'end of the probe P1-1 (SEQ ID NO: 1), namely, the primer anchoring regions (A'), are reversely complementary.

(2) A single PCR assay was performed on Respiratory Syncytial Virus (RSV) using the primer probes described above.

The reaction system used in the detection is shown in Table 2.

Table 2: respiratory syncytial virus single PCR detection reaction system

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Note that: the 2 XPCR reaction buffer comprises 3mM MgCl ₂ 30mM Tris-HCl buffer, pH 8.3, 0.5mM dNTP and 70mM (NH) ₄ ) ₂ SO ₄ 。

Nucleic acid templates: the in vitro transcribed RNA of the target sequence of the respiratory syncytial virus is used as a nucleic acid template, and the target sequence of the respiratory syncytial virus is separately detected by using reverse primers (R) with different signal detection region sequences, so as to verify the detection capability of single virus infection. Pure water was used as a no-template control (NTC).

And (3) preparing the following reaction:

after the preparation of the nucleic acid template, the reaction system was prepared in accordance with the ratio shown in Table 2.

PCR amplification and melting curve analysis:

the PCR tube was capped, gently mixed, centrifuged briefly and allowed to stand at room temperature for 5min. The PCR tube was again placed in a palm centrifuge and after brief centrifugation transferred to a tray of Shanghai Marble medical technology Co., ltd., SLAN real-time fluorescent quantitative PCR instrument. The procedure used was: reverse transcription at 55℃for 15 min; pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 10 seconds, annealing at 56 ℃ for 30 seconds, 45 cycles in total, and no lighting; denaturation at 94℃for 150 seconds, annealing at 40℃and extension for 300 seconds; analyzing a dissolution curve at 40-85 ℃, heating to 0.05 ℃ per second, and lighting to obtain a dissolution curve.

Data analysis:

and judging the result by selecting SLAN real-time fluorescence quantitative PCR instrument analysis software of Shanghai macro stone medical science and technology company through the Tm value of the melting curve. FIG. 1 is a melting graph of a positive sample of a single PCR respiratory syncytial virus amplified by a reverse primer (R) with a signal detection region.

FIG. 1 shows that PCR wells added with RSV positive template formed melting peaks in the CY5 channel with Tm of 71.64 ℃. The design method of the primer probe can be used for clearly detecting the target by adopting PCR amplification and melting curve analysis.

Example 2: single PCR detection of Respiratory Syncytial Virus (RSV)

(1) The base sequences of primer probes for detecting Respiratory Syncytial Virus (RSV) by multiplex PCR are shown in table 3.

Table 3: base sequence of primer probe

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Among the above primer probes:

the first sequence (SEQ ID NO: 3) of the forward primer F1-1 is a specific primer designed for a target sequence of respiratory syncytial virus, the total length of the first sequence of the forward primer F1-1 is 44bp, the 1 st to 29 th bases at the 3 'end of the forward primer are the target sequence binding region 1, and the 1 st to 12 th bases at the 5' end of the forward primer are the amplification product capturing region (B);

the first sequence (SEQ ID NO: 6) of the reverse primer R1-2 is a specific primer designed for a target sequence of respiratory syncytial virus, the total length of the first sequence of R1-2 is 40bp, the 1 st to 24 th bases at the 3 'end of the first sequence are the target sequence binding region 2, and the 1 st to 13 th bases at the 5' end of the first sequence are the reverse amplification product capturing region (C);

the second sequence (SEQ ID NO: 7) of the forward primer F1-2 has a total length of 25bp, the 1 st to 12 th bases at the 3 'end thereof are amplification product capturing regions (B), the 1 st to 10 th bases at the 5' end thereof are probe anchoring regions (A), and the 1 st to 10 th base sequences at the 5 'end of the probe P1-2 (SEQ ID NO: 5), namely, the primer anchoring regions (A'), are reversely complementary;

The second sequence (SEQ ID NO: 8) of the reverse primer R1-2 has a total length of 24bp, the 1 st to 8 th bases at the 5 'end thereof are primer signal detection regions (h), and the 1 st to 13 th bases at the 3' end thereof are reverse amplification product capture regions (C).

The reaction system used in the detection is shown in Table 4.

Table 4: respiratory syncytial virus single PCR detection reaction system

Reagent component	Concentration of
		2 XPCR reaction buffer	1×
DNA polymerase	2U
		Reverse transcriptase	5U
Probe (P1-2)	400nM
		First sequence of forward primer (F1-1)	100nM
First sequence of reverse primer (R1-2)	45nM
		Second sequence of forward primer (F1-2)	250nM
Second sequence of reverse primer (R1-2)	100nM
		Nucleic acid templates	30 copies
Ultrapure water	To 20. Mu.L

Nucleic acid templates: in vitro transcribed RNA of the respiratory syncytial virus target sequence was used as a nucleic acid template. Pure water was used as a no-template control (NTC).

And (3) preparing the following reaction:

after the preparation of the nucleic acid template, the reaction system was prepared in accordance with the proportions shown in Table 4.

PCR amplification and melting curve analysis:

the PCR tube was capped, gently mixed, centrifuged briefly and allowed to stand at room temperature for 5min. The PCR tube was again placed in a palm centrifuge and after brief centrifugation transferred to a tray of Shanghai Marble medical technology Co., ltd., SLAN real-time fluorescent quantitative PCR instrument. The procedure used was: reverse transcription at 55℃for 15 min; pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 10 seconds, annealing at 56 ℃ for 30 seconds, 45 cycles in total, and no lighting; denaturation at 94℃for 150 seconds and annealing at 40℃for 300 seconds; and (3) analyzing a dissolution curve at 40-85 ℃, wherein the heating rate is 0.05 ℃ per second, and lighting to obtain a dissolution curve, as shown in figure 2.

Data analysis:

and judging the result by selecting SLAN real-time fluorescence quantitative PCR instrument analysis software of Shanghai macro stone medical science and technology company through the Tm value of the melting curve.

FIG. 2 shows that PCR wells added with RSV-positive template form melting peaks in the VIC channel with Tm of 82.32 ℃. The design method of the primer probe can be used for clearly detecting the target by adopting PCR amplification and melting curve analysis.

It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.

SEQUENCE LISTING

<110> Mike organism Co., ltd

<120> a primer probe for detection, primer probe set, and use thereof

<130> 2021

<160> 8

<170> PatentIn version 3.3

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<210> 7

<211> 25

<212> DNA

<213> second sequence of F1-2 (Artificial sequence)

<400> 7

gctcccaggg aaagactgca gtcaa 25

<210> 8

<211> 24

<212> DNA

<213> second sequence of R1-2 (artificial sequence)

<400> 8

cggcaccgaa acgcattaca agtt 24

Claims

1. A primer probe for PCR detection, comprising a forward primer (F), a probe (P) and a reverse primer (R); it is characterized in that the method comprises the steps of,

2. The primer probe according to claim 1, wherein the forward primer (F) consists of two separate sequences and the reverse primer (R) consists of one separate sequence;

3. The primer probe of claim 2, wherein the second sequence of the forward primer (F) comprises, in order from the 5 'end to the 3' end, a probe anchor region (a) and an amplified product capture region (B);

4. The primer probe according to claim 1, wherein the forward primer (F) and the reverse primer (R) are each composed of two separate sequences;

the first sequence of the reverse primer (R) sequentially comprises a reverse amplification product capturing region (C) and a target sequence binding region 2 from the 5 'end to the 3' end, the second sequence of the reverse primer (R) sequentially comprises a primer signal detecting region (h) and a reverse amplification product capturing region (C) from the 5 'end to the 3' end, and the reverse amplification product capturing region (C) is a sequence spacer which is not in pairing combination with any target sequence.

5. The primer probe of claim 4, wherein the second sequence of the forward primer (F) comprises, in order from the 5 'end to the 3' end, a probe anchor region (a) and an amplified product capture region (B);

6. The primer probe of any one of claims 1-5, wherein the detection moiety comprises a first detection moiety and a second detection moiety, and wherein the first detection moiety and the second detection moiety produce a change in signal by a change in distance; preferably, the first detection group and the second detection group are separated by 5-25 bases; further preferably, the first detection moiety is a fluorescent reporter moiety and the second detection moiety is a quencher moiety or other modifying moiety capable of generating a signal change from the first detection moiety via fluorescence resonance energy transfer.

7. The primer-probe according to claim 3 or 5, characterized in that the probe (P) comprises, in order from the 5' end to the 3' end, a primer-anchor region (a ') and a probe signal detection region (H); preferably, the detection group on the probe (P) is modified between the primer anchor region (A') and the probe signal detection region (H).

8. The primer-probe according to claim 3 or 5, characterized in that the probe (P) comprises, in order from the 5' end to the 3' end, a probe signal detection region (H) and a primer-anchor region (a '); preferably, the detection group on the probe (P) is modified at the 5' end of the probe signal detection zone (H).

9. The primer probe of any one of claims 1 to 8, wherein the 3' end of the probe (P) contains a blocking region.

10. The primer-probe according to any one of claims 1 to 9, wherein the Tm value of the primer-signal detection region (h) is simultaneously smaller than the Tm value of the target sequence-binding region 1 in the forward primer (F) and the Tm value of the target sequence-binding region 2 in the reverse primer (R).

11. The primer-probe according to claim 10, wherein the Tm value of the primer-anchor region (A') is larger than the Tm value of the primer-signal detection region (h).

12. A kit for PCR detection comprising the primer probe for PCR detection of any one of claims 1 to 11; preferably, further comprising an amplification reagent; further preferably, the amplification reagents comprise DNA polymerase and dNTPs; still further preferably, when the nucleic acid template is RNA, the amplification reagent further comprises reverse transcriptase.

13. A primer probe set for PCR detection comprising at least 1 probe (P), at least 2 forward primers (F) and at least 2 reverse primers (R); it is characterized in that the method comprises the steps of,

wherein at least 1 forward primer (F) and/or its corresponding reverse primer (R) consists of two separate sequences;

14. The probe primer set according to claim 13, wherein the at least 1 forward primer (F) consists of two separate sequences, and the reverse primer (R) corresponding to the forward primer (F) consists of one separate sequence;

15. The primer probe set of claim 14, wherein the second sequence of the at least 1 forward primer (F) comprises, in order from the 5 'end to the 3' end, a probe anchor region (a) and an amplification product capture region (B);

the probe (P) is a sequence which is not in pairing connection with any target sequence and comprises a primer anchor region (A') and a probe signal detection region (H); the primer anchor region (A') is a sequence complementary to the probe anchor region (A); preferably, the primer anchor region (A') and/or the probe signal detection region (H) of different said probes (P) differ in sequence and the modified detection groups differ.

16. Primer probe set according to claim 13, characterized in that the at least 1 forward primer (F) and its corresponding reverse primer (R) consist of two separate sequences;

17. The primer probe set of claim 16, wherein the second sequence of the at least 1 forward primer (F) comprises, in order from the 5 'end to the 3' end, a probe anchor region (a) and an amplification product capture region (B);

18. The primer probe set of any one of claims 13-17, wherein the detection moiety comprises a first detection moiety and a second detection moiety, and wherein the first detection moiety and the second detection moiety produce a change in signal by a change in distance; preferably, the first detection group and the second detection group are separated by 5-25 bases; further preferably, the first detection moiety is a fluorescent reporter moiety and the second detection moiety is a quencher moiety or other modifying moiety capable of generating a signal change from the first detection moiety via fluorescence resonance energy transfer.

19. The set of primer probes according to any one of claims 13-18, wherein the 3' -end of the probe (P) comprises a blocking region.

20. The primer-probe set of any one of claims 13-19, wherein the Tm value of the primer-signal detection region (h) is simultaneously less than the Tm value of the target sequence-binding region 1 in the forward primer (F) and the Tm value of the target sequence-binding region 2 in the reverse primer (R).

21. The primer-probe set of claim 120, wherein the Tm value of the primer-anchor region (a') is greater than the Tm value of the primer-signal detection region (h).

22. A kit for PCR detection comprising the primer probe set for PCR detection of any one of claims 13 to 21; preferably, further comprising an amplification reagent; further preferably, the amplification reagents comprise DNA polymerase and dNTPs; still further preferably, when the nucleic acid template is RNA, the amplification reagent further comprises reverse transcriptase.

23. A method of PCR detection of a sample to be detected using the kit of claim 12 or 22, comprising the steps of:

s3, carrying out PCR amplification on the reaction system;

s4, analyzing whether the target object exists.

24. The method according to claim 23, wherein said step S4 comprises the steps of:

25. A method of PCR detection of a sample to be detected using the kit of claim 12 or 22, comprising the steps of:

26. The method according to claim 24 or 25, wherein when the obtained Tm value of the peak position of the melting curve is within the range of the Tm value of the target in the sample to be tested, which is characteristic of the specific detection channel, it indicates that the sample to be tested contains the target; preferably, the specific detection channel is a fluorescent channel corresponding to the first detection group on the probe (P), and the characteristic Tm value is a melting temperature of a double-strand product of the secondary amplification formed by the probe (P).