CN116875710A

CN116875710A - Construction method of amplification system for identifying mycoplasma and primer probe combination thereof

Info

Publication number: CN116875710A
Application number: CN202310403122.4A
Authority: CN
Inventors: 柳丽萍; 韩晋; 郭求真
Original assignee: Rocgene Tecnology Co
Current assignee: Rocgene Tecnology Co
Priority date: 2023-04-14
Filing date: 2023-04-14
Publication date: 2023-10-13

Abstract

The invention provides a construction method of an amplification system for identifying mycoplasma and a primer probe combination thereof, and the construction method of the amplification system for identifying mycoplasma comprises the following steps: designing a primer and a probe; wherein the primer and the probe comprise: a consensus sequence; screening the primers and the probes; wherein, the primer obtained by screening has the characteristic of consistent specificity of the amplified product; respectively constructing a single amplification system; wherein each of said single amplification systems corresponds to at least one mycoplasma microorganism; the probes in each single amplification system have a matching relationship with the primers; combining and combining at least two single amplification systems corresponding to different mycoplasma microorganisms to construct a multiplex amplification system.

Description

Construction method of amplification system for identifying mycoplasma and primer probe combination thereof

Technical Field

The invention relates to the technical field of biological genes, in particular to a construction method of an amplification system for identifying mycoplasma and a primer probe combination thereof.

Background

Mycoplasma is the smallest and simplest self-replicating organism. They vary in size from 0.2 to 0.8 μm and can pass through some filters for bacterial removal; their genome sizes range from about 540kb to 1300kb with G+C content of 23 to 41%. Although mycoplasma originates from gram-positive bacterial branches of walled bacteria, evolution results in a significant decrease in genome size, losing the functions required for synthesis and maintenance of bacterial cell walls. Mycoplasma have no cell wall, so that antibiotics that normally act on the cell wall do not act on it.

Mycoplasma is a common bacterial contaminant in cell culture samples. Mycoplasma in infected cell cultures can alter many cellular processes, including altering cell growth rates, inducing morphological changes or cell transformations, and mimicking viral infections. According to the regulatory requirements of Chinese pharmacopoeia and NMPA (national drug administration), cell culture in drug production must be free of mycoplasma. Therefore, there is a need for routine, periodic detection of possible contamination of all cell cultures used in pharmaceutical production.

Existing methods for identifying mycoplasma contamination have relied primarily on traditional bacterial culture on agarose plates. Because mycoplasma grow slowly (colonies may take up to 3 weeks to form), traditional growth-based methods require at least 28 days of incubation to definitively exclude contamination. The sampling and result interval is too long, preventing effective Quality Control (QC) inspection, delaying production approval, limiting the sampling points of QC. In contrast, nucleic acid amplification technology (NAT) can shorten the time to obtain results to several hours. In addition, NAT can also detect different mycoplasma simultaneously.

In chinese pharmacopoeia 2020 edition, "examination of mycoplasma 3301", it is indicated that "other methods approved by the national drug testing agency may be used in addition to culture and indicator cell culture (DNA staining). The national enterprises generally refer to the NAT method of European pharmacopoeia or Japanese pharmacopoeia to verify at present. As an alternative to the culture method, the NAT test system is capable of detecting 10CFU/mL mycoplasma.

Disclosure of Invention

The invention provides a construction method of an amplification system for identifying mycoplasma and a primer probe combination thereof, which are used for solving the problem of detecting and identifying whether mycoplasma exists in a sample.

The construction method of the amplification system for identifying mycoplasma provided by the invention comprises the following steps:

designing a primer and a probe; wherein the primer and the probe comprise: a consensus sequence; wherein the consensus sequence is a consensus sequence capable of covering at least two mycoplasma microorganisms;

screening the primers and the probes; wherein, the primer obtained by screening has the characteristic of consistent specificity of the amplified product;

respectively constructing a single amplification system; wherein each of said single amplification systems corresponds to at least one mycoplasma microorganism; the probes in each single amplification system have a matching relationship with the primers;

combining and merging at least two single amplification systems corresponding to different mycoplasma microorganisms to construct a multiple amplification system;

wherein the primers of the multiplex amplification system comprise: a first sequence combination; the first sequence combination includes: the primers of the single amplification system comprise the consensus sequence;

Wherein the probes of the multiplex amplification system comprise: a second sequence combination; the second sequence combination includes: the probes of the single amplification system comprise the consensus sequence.

Further, the method for constructing an amplification system for identifying mycoplasma according to the present invention, the step of designing a primer and a probe includes:

establishing a common sequence search region of mycoplasma microorganism;

the consensus sequence search region searches for a consensus sequence capable of covering at least one of Zhi Yuanti, spiroplasmataceae and cholesteryl-free families.

Further, the method for constructing an amplification system for identifying mycoplasma according to the present invention comprises selecting the consensus sequence in the consensus sequence search region according to at least one of the following criteria:

the length of the consensus sequence is more than or equal to 30bp;

no more than 3 discrete degenerate bases of the consensus sequence;

the degenerate bases of the consensus sequence are located 5' to the consensus sequence.

Further, the method for constructing an amplification system for identifying mycoplasma according to the present invention, wherein the consensus sequence is capable of covering at least one mycoplasma microorganism selected from the group consisting of:

Mycoplasma hyorhinis；

Mycoplasma arginini；

Mycoplasma gallisepticum；

Mycoplasma pneumoniae；

Acholeplasma laidiawii；

Mycoplasma fermentans；

Spiroplasma citri；

Mycoplasma synoviae；

Mycoplasma orale；

Mycoplasma salivarium。

further, the method for constructing an amplification system for identifying mycoplasma according to the present invention, wherein the steps of screening the primer and the probe include:

Performing an amplification reaction including a melting curve stage on the designed primer to obtain a melting curve and an amplification curve; wherein, the plasmid template that adopts includes: a high concentration plasmid template and a low concentration plasmid template;

screening primers based on the obtained melting curve and amplification curve to obtain primers with consistent specificity of amplification products;

based on the primers obtained by the screening, the probes are screened.

Further, the method for constructing an amplification system for identifying mycoplasma according to the present invention, wherein the steps of constructing a single amplification system respectively include:

optimizing the primer concentration of each of the single amplification systems;

optimizing the annealing temperature of each single amplification system;

optimizing the probe concentration of each single amplification system based on the primer concentration and the annealing temperature obtained by optimization;

optimizing an amplification system of each of the single amplification systems based on the primer concentration, the annealing temperature, and the probe concentration obtained by the optimization;

wherein the optimization of the amplification system of the single amplification system comprises at least one of:

selection of an amplification enzyme;

selecting a buffer solution;

the amount of dNTPs used;

mg ion concentration.

Further, the method for constructing an amplification system for identifying mycoplasma according to the present invention, wherein the step of combining and combining the single amplification systems of at least two mycoplasma microorganisms of different species to construct a multiplex amplification system comprises:

combining the preliminary single amplification systems by taking the groups as units to obtain multiple amplification systems of each group;

detecting, screening and optimizing each group of multiplex amplification systems by adopting the reaction conditions of the single amplification systems in the groups;

wherein the single amplification system combination criteria comprises at least one of:

avoiding the generation of dimers between primers or probes of different pooled single amplification systems;

the single amplification systems that can share primers or probes are combined in the same set;

combining the single amplification systems with the difference value of the amplification reaction conditions within a preset difference value range into the same group;

single amplification systems with repeated detection functions are excluded.

Further, the method for constructing an amplification system for identifying mycoplasma according to the present invention, after the step of constructing a multiplex amplification system, further comprises:

setting internal reference detection;

wherein, the selection criteria of the primer and the probe for the internal reference detection include:

The primer and the probe for internal reference detection adopt sequences different from those of mycoplasma microorganism;

the primer and the probe for internal reference detection and the primer and the probe of the multiplex amplification system do not generate dimers, and the difference value between the Tm values of the primer and the probe of the multiplex amplification system is smaller than a preset threshold value;

the difference between the amplification reaction conditions of the internal reference detection and the amplification reaction conditions of the multiplex amplification system is within a preset difference range.

Further, the method for constructing the amplification system for identifying mycoplasma, disclosed by the invention, comprises the following steps of:

(d1) SEQ ID NO. 67 or SEQ ID NO. 68;

(d2) A sequence shown in SEQ ID NO. 67 or SEQ ID NO. 68 is subjected to substitution, deletion or addition of one or more nucleotides and keeps the specificity of the amplified products consistent;

the probes for reference detection include the following sequences:

(d3) The sequence shown in SEQ ID NO. 69;

(d4) The sequence shown in SEQ ID NO. 69 is a sequence with identical specificity of amplified products by substituting, deleting or adding one or more nucleotides.

Further, the method for constructing the amplification system for identifying mycoplasma, disclosed by the invention, comprises the following common sequences:

(a1) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 44 or the sequence shown in SEQ ID NO. 43, 42, 44;

alternatively, the primers of the single amplification system comprise the following consensus sequences:

(a2) SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 42, SEQ ID NO. or SEQ ID NO. 42;

(b1) SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, or a sequence shown as SEQ ID NO 66;

alternatively, the probes of the single amplification system comprise the following consensus sequences:

(b2) SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, or SEQ ID NO 66 are substituted, deleted, or added with one or more nucleotides and maintain the sequence of the amplified products in specific agreement;

Wherein the primers in the same single amplification system are matched with the probes to ensure that the amplification reaction with consistent specificity of the primer amplification products can be performed.

Further, the method for constructing an amplification system for identifying mycoplasma according to the present invention, wherein the first sequence combination of the primers of the multiplex amplification system comprises:

(c1) SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 13, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 38 and SEQ ID NO. 42;

alternatively, the first sequence combination of primers of the multiplex amplification system comprises:

(c2) SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 13, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 38 and SEQ ID NO. 42 are substituted, deleted or added with one or more nucleotides and keep the specificity of the amplified products consistent;

the second sequence combination of probes of the multiplex amplification system comprises:

(c3) SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 62 and SEQ ID NO. 66;

Alternatively, the second sequence combination of probes of the multiplex amplification system comprises:

(c4) SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 62 and SEQ ID NO. 66 are substituted, deleted or added with one or several nucleotides and keep the specificity of the amplified products consistent.

In an alternative embodiment of the present invention, the method for constructing an amplification system for identifying mycoplasma according to the present invention, wherein the first sequence combination of primers of the multiplex amplification system comprises:

the first sequence combination of primers of the multiplex amplification system comprises:

(c5) SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 13, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 and SEQ ID NO. 38;

(c6) SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 13, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 and SEQ ID NO. 38 are substituted, deleted or added with one or more nucleotides and keep the specificity of the amplified products consistent;

(c7) SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 60 and SEQ ID NO. 66;

(c8) SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 60 and SEQ ID NO. 66 are substituted, deleted or added with one or several nucleotides and keep the specificity of the amplified products consistent.

The invention provides a primer probe combination for identifying mycoplasma, which comprises the following primers: a first sequence combination; the first sequence combination includes: at least one consensus sequence;

the probe comprises: a second sequence combination; the second sequence combination includes: at least one consensus sequence;

the consensus sequence employs a consensus sequence capable of covering at least two mycoplasma microorganisms.

Further, the primer probe combination for identifying mycoplasma, provided by the invention, can cover at least one of Zhi Yuanti families, spiroplasmaceae and cholesteryl-free families;

the first sequence combination and the second sequence combination are capable of covering all three of Zhi Yuanti, spiroplasmataceae and cholesteryl-free families;

The consensus sequence satisfies at least one of the following conditions:

the length of the consensus sequence is more than or equal to 30bp;

no more than 3 discrete degenerate bases of the consensus sequence;

Further, the primer probe combination for identifying mycoplasma, the primer of the first sequence combination and the probe of the second sequence combination belong to a multiplex amplification system;

the multiplex amplification system is obtained by combining and combining at least two single amplification systems corresponding to different mycoplasma microorganisms;

wherein each of said single amplification systems corresponds to at least one mycoplasma microorganism; the probes within each of the multiplex amplification systems have a matching relationship with the primers.

Further, the primer probe combination for identifying mycoplasma in the invention, the single amplification system corresponds to at least one mycoplasma microorganism of the following:

Mycoplasma hyorhinis；

Mycoplasma arginini；

Mycoplasma gallisepticum；

Mycoplasma pneumoniae；

Acholeplasma laidiawii；

Mycoplasma fermentans；

Spiroplasma citri；

Mycoplasma synoviae；

Mycoplasma orale；

Mycoplasma salivarium；

the multiplex amplification system is capable of covering all mycoplasma microorganisms:

Mycoplasma hyorhinis；

Mycoplasma arginini；

Mycoplasma gallisepticum；

Mycoplasma pneumoniae；

Acholeplasma laidiawii；

Mycoplasma fermentans；

Spiroplasma citri；

Mycoplasma synoviae；

Mycoplasma orale；

Mycoplasma salivarium。

furthermore, the primer probe combination for identifying mycoplasma,

the consensus sequence of the primer comprises:

(a1) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 44 or 43 of the sequence shown in SEQ ID NO 1, 17, 33, 34, 40, 42, or 43.

Alternatively, the consensus sequence of the primer comprises:

(a2) SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 44, SEQ ID NO. 43, SEQ ID NO. 42, SEQ ID NO. 44;

the consensus sequence of the probe comprises:

Alternatively, the consensus sequence of the probe comprises:

and the primer in the same single amplification system is matched with the probe so as to ensure that the amplification reaction with consistent specificity of the primer amplification product can be carried out.

Furthermore, the primer probe combination for identifying mycoplasma,

the first sequence combination of primers comprises:

alternatively, the first sequence combination of primers comprises:

The second sequence combination of probes comprises:

alternatively, the second sequence combination of probes comprises:

(c4) Sequences shown in SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 62 and SEQ ID NO. 66 are substituted, deleted or added with one or more nucleotides and keep the specificity of amplified products consistent;

in an alternative embodiment of the present invention, the primer probe combination for identifying mycoplasma according to the present invention,

the first sequence combination of primers comprises:

alternatively, the first sequence combination of primers comprises:

The second sequence combination of probes comprises:

alternatively, the second sequence combination of probes comprises:

The invention also provides a multiplex amplification system for identifying mycoplasma, which adopts the primer probe combination.

Further, the multiplex amplification system for identifying mycoplasma according to the present invention further comprises: detecting internal parameters;

the primer for internal reference detection comprises the following sequences:

(d1) SEQ ID NO. 67 or SEQ ID NO. 68;

the probes for reference detection include the following sequences:

(d3) The sequence shown in SEQ ID NO. 69;

Further, the multiplex amplification system for identifying mycoplasma according to the present invention has the following reaction conditions:

the primer concentrations included: 100 to 150nM;

the annealing temperature includes: 55 to 65 ℃;

probe concentration includes; 50 to 100nM;

amplification enzymes include, but are not limited to: uracil-DNA glycosylase, DNA polymerase; wherein the DNA polymerase comprises: taq enzyme, high fidelity enzyme, or hot start enzyme;

buffers include, but are not limited to: tris hydrochloride buffer, potassium chloride, magnesium sulfate, bovine Serum Albumin (BSA), glycerol;

dNTPs include: 100. Mu.M dATP, 100. Mu.M dGTP, 100. Mu.M dTTP, 100. Mu.M dCTP, 10. Mu.M dUTP;

the concentration of Mg ions includes: 3.2mM magnesium ion.

The invention also provides a kit for identifying mycoplasma, which adopts the primer probe combination.

The invention also provides another kit for identifying mycoplasma, which adopts the multiplex amplification system.

The invention also provides a kit for identifying mycoplasma, which adopts a multiplex amplification system;

the multiplex amplification system is obtained by adopting the construction method of the amplification system for identifying mycoplasma.

According to the construction method of the amplification system for identifying mycoplasma and the primer probe combination thereof, provided by the invention, a multiplex qPCR detection system is established based on the consensus sequence, so that the most simplified primer probe combination can be used for covering the microorganisms of mycoplasma as many as possible.

Based on the specific technical scheme provided by the embodiment of the invention, the qualitative detection of more than 150 mycoplasma microorganisms (mycoplasma family, non-cholesteroliaceae, spirochete) can be realized, wherein the qualitative detection comprises 10 important mycoplasma required to be detected in European pharmacopoeia and Japanese pharmacopoeia. The detection system has high specificity and no cross reaction to bacteria and mammal DNA. The sensitivity of the invention can reach 10CFU/mL by matching with mycoplasma nucleic acid extraction reagent. The test is verified by referring to European pharmacopoeia EP 2.6.7 and Japanese pharmacopoeia JP XVII mycoplasma test-related requirements, and mycoplasma test can be performed instead of cell culture methods.

Drawings

Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, made with reference to the accompanying drawings in which:

FIG. 1 is a schematic flow chart of a method for constructing an amplification system for identifying Mycoplasma according to the first embodiment of the present invention;

FIG. 2 is a graph of qPCR results of Lei cholesterol free minimum detection Limit (LOD) according to an embodiment of the present invention;

FIG. 3 is a graph of qPCR results of the minimum limit of detection (LOD) of Mycoplasma fermentum of the present invention;

FIG. 4 is a graph of qPCR results of minimum limit of detection (LOD) of Mycoplasma gallisepticum in accordance with the embodiments of the present invention;

FIG. 5 is a graph of qPCR results of the minimum limit of detection (LOD) of Mycoplasma hyopneumoniae of the present invention;

FIG. 6 is a graph of qPCR results for minimum detection Limit (LOD) of Mycoplasma stomatitis in accordance with an embodiment of the present invention;

FIG. 7 is a graph of qPCR results for the minimum limit of detection (LOD) of Mycoplasma pneumoniae of the present invention;

FIG. 8 is a graph of qPCR results of minimum limit of detection (LOD) of Mycoplasma synoviae according to an embodiment of the present invention;

FIG. 9 is a graph of qPCR results of the minimum limit of detection (LOD) of Mycoplasma arginine according to an embodiment of the invention;

FIG. 10 is a graph of qPCR results of minimum detection Limit (LOD) of Mycoplasma salivarius according to an embodiment of the present invention;

FIG. 11 is a graph of qPCR results of the lowest limit of detection (LOD) of the helicobacter mandarin in an embodiment of the present invention;

FIG. 12 is a graph showing the results of DNA amplification of ten bacteria and fungi according to the example of the present invention;

FIG. 13 is a graph showing the results of DNA amplification in four mammals according to the embodiment of the present invention.

Detailed Description

The following detailed description of the invention, given by way of illustration only and not limitation, will be readily apparent to those skilled in the art in view of the present disclosure.

Example 1

FIG. 1 is a schematic flow chart of a method for constructing an amplification system for identifying mycoplasma according to an embodiment of the present invention, as shown in FIG. 1, the method for constructing an amplification system for identifying mycoplasma provided by the present invention includes:

step S101, designing a primer and a probe; wherein the primer and the probe comprise: a consensus sequence; wherein the consensus sequence is a consensus sequence capable of covering at least two mycoplasma microorganisms.

Among others, the aim of the invention is to detect the presence of mycoplasma with as compact a primer probe combination as possible, thus using a consensus sequence to cover as many mycoplasma microorganisms as possible. The consensus sequence refers to the same sequence segment of different species of microorganism.

Step S102, screening the primers and the probes; wherein, the primer obtained by screening has the characteristic of consistent specificity of amplified products.

Wherein, the primer is used for obtaining the specific consistent amplification product through an amplification reaction so as to detect whether the sample contains a target sequence. It is generally considered that the consistency of the fragment sequences of the amplified products is greater than 90%, and that the amplified products are consistent in specificity. The primer sequence is designed through the step 101, and whether the primer sequence can meet the characteristic of consistent specificity of amplified products is further detected.

Step S103, respectively constructing a single amplification system; wherein each of said single amplification systems corresponds to at least one mycoplasma microorganism; the probes within each of the multiplex amplification systems have a matching relationship with the primers.

The primer probe sequence obtained in the step S102 is used as a part of a single amplification system, and then various reaction conditions such as primer concentration, annealing temperature, probe concentration and the like of the single amplification system are optimized to obtain a complete single amplification system capable of detecting a mycoplasma microorganism or a plurality of mycoplasma microorganisms.

Step S104, combining and combining at least two single amplification systems corresponding to different mycoplasma microorganisms to construct a multiple amplification system.

Wherein, based on similar reaction conditions, a plurality of single amplification systems are combined to form a complete multiplex amplification system, so that the multiplex amplification system can cover as many mycoplasma microorganism species as possible.

Wherein the primer sequences of the multiplex amplification system are essentially a sequence combination of primer sequences of several pooled single amplification systems. Such a combination of sequences may be a combination of several consensus sequences or a combination of consensus sequences and non-consensus sequences. However, for the present invention, the sequence combination needs to include a consensus sequence in order to reduce the primer combination as much as possible. However, the incorporation of a single amplification system of non-consensus sequences into the multiplex amplification system of the present invention is employed such that the combination of sequences includes non-consensus sequences in addition to consensus sequences, and still forms part of the inventive concept, as alternatives readily occur to those skilled in the art.

Steps S101 to S104 are specifically described below.

Specifically, the designing primer and probe in step S101 includes: steps S1011 to S1012.

In step S1011, a consensus search region of Mycoplasma microorganisms is established.

Step S1012, searching the consensus sequence search region for a consensus sequence capable of covering at least one of Zhi Yuanti, spiroplasmataceae and cholesteryl-free families.

Specifically, the 16S rRNA coding region (i.e., 16S ribosomal RNA) of hundreds of Mycoplasma microorganisms (emphasis includes Mycoplasma, cholesterol, spirulina) was searched. The consensus sequences of these microorganisms are searched and labeled to create a consensus search region. Primers and probes are designed in the common sequence search area so as to cover as many mycoplasma microorganisms as possible by the most simplified primer probe combination. The search and design software can select public biological tools such as NCBI-blast, clustalX, vector NTI Advance11, DNAman and the like, or use an autonomously developed sequence alignment program.

In actual practice, there is no consensus sequence covering all target microorganisms; different consensus sequences cover different ranges of target microorganisms, often the longer the consensus sequence, the smaller the range of microorganisms covered. The sequence difference among the three is larger.

For the above case, the positions of the primer probes were determined according to the following 4 criteria:

1. the method comprises the steps of respectively comparing the consensus sequences of the non-cholesterin mycoplasma, the spiroplasma and the mycoplasma, and respectively designing respective primer probes;

2. searching for a region of a consensus sequence of other Mycoplasma order microorganisms with 8 Mycoplasma, 1 spiroplasma and 1 cholesteryl-free mycoplasma in Table 1 below;

3. The length of the consensus sequence is greater than 30bp, so that enough regions are provided for designing primers or probes;

4. the consensus sequence may have degenerate bases present, but discontinuous degenerate bases typically do not exceed 3.

Numbering device	Strain name	Attribution to
			1	Acholeplasma laidlawii	No cholest
2	Mycoplasma arginini	Mycoplasma species
			3	Mycoplasma fermentans	Mycoplasma species
4	Mycoplasma gallisepticum	Mycoplasma species
			5	Mycoplasma hyorhinis	Mycoplasma species
6	Mycoplasma orale	Mycoplasma species
			7	Mycoplasma pneumoniae	Mycoplasma species
8	Mycoplasma salivarium	Mycoplasma species
			9	Mycoplasma synoviae	Mycoplasma species
10	Spiroplasma citri	Spiromelania species

Table 1 european pharmacopoeia and japanese pharmacopoeia prescribe common mycoplasma causing cell contamination to be detected

Specifically, primer/probe design can be performed using design software such as primer 5,Oligo,SnapGene,Primer Express. In addition to the design suggestions recommended by the design software, the design of the primers/probes also avoids the positions of degenerate bases in the consensus sequence as much as possible. In the case where degenerate bases must be used, they are located as far as possible at the 5' end of the sequence. After the primer probe sequence design is completed, the synthesis is ordered, and the synthesis can be ordered by a biological engineering (Shanghai) stock company. The probes can be uniformly labeled by FAM fluorescence, and the quenching groups can be MGB or BHQ1 quenching groups according to the types of the probes. In addition, it is necessary to order plasmids containing different target sequences for screening, optimization, etc. of an amplification system.

In particular, all probes may be fluorescently labeled with the same probe. Because mycoplasma (spiroplasma, non-cholesterolplasma) contamination is difficult to remove, in practice, if mycoplasma is contaminated, cells or biological products are generally selected to be directly discarded, and it is not significant to distinguish which mycoplasma is specific to cause contamination. If the cells or cultures and their precious nature require a clear distinction between the sources of contamination, other methods may be used for further identification, such as sequencing of the amplified products. Such cases are not included in the intended scope of use of the present invention.

Specifically, the screening of the primers and probes in step S102 includes: steps S1021 to S1023.

S1021, performing amplification reaction comprising a melting curve stage on the designed primer to obtain a melting curve and an amplification curve; wherein, the plasmid template that adopts includes: a high concentration plasmid template and a low concentration plasmid template;

s1022, screening primers based on the obtained melting curve and amplification curve to obtain primers with consistent specificity of amplification products;

s1023, screening the probes based on the primers obtained by screening.

Specifically, the synthesized primers and probes are dissolved and diluted to appropriate concentrations: the stock solution for the primers and probes was typically 100. Mu.M, and the working solution was 10. Mu.M. The synthesized plasmid dry powder is generally dissolved by using 200-500 mu L of TE buffer (pH 8.0) or other nucleic acid preservation solution, and is packaged and preserved; diluted with nuclease-free water and used.

Specifically, primer screening was performed. The primers are paired according to different combinations, a SYBR Green dye method is used for amplifying a single primer pair (comprising a melting curve stage), and a plasmid template is selected to be at a high concentration or a low concentration, wherein the concentration of the template is preferably 100-200 copies/. Mu.L. The reaction system refers to the instruction of the used amplification enzyme reagent product; primary test primer concentrations were selected between 100-200 nM; an example of the running program can refer to table 2, and parameters can be adjusted according to actual conditions.

Table 2 run program example: archimed X4 fluorescent quantitative PCR instrument

Specifically, after the melting curve and amplification curve are obtained, the primers can be selected according to the following 3 criteria:

1. the melting curve of the high-concentration template has single characteristic peak of the target, and the Ct value of the amplification curve is lower than a preset threshold A; ct value (cycle threshold) refers to the number of amplification cycles corresponding to the time when the fluorescence signal of the amplified product reaches the set fluorescence threshold in the amplification process of fluorescence quantitative PCR;

2. melting curve of low concentration template, obvious target peak, no or lower non-target peak;

3. the amplification curve of NTC is signal-free; NTC (No template control) refers to a blank.

After the completion of the primer selection, probe selection is performed using the preferred primers. Adding matched primer and probe into the amplification system, and selecting the concentration of the primary test probe to be 100-200nM, wherein the concentration of the primer is the same as that of the primer screening step. The amplification system does not contain SYBR Green dye, and the running program refers to a primer screening step and removes a melting curve stage. Plasmid templates use both high and low concentration gradients, or only low concentrations are selected.

Specifically, probes can be screened according to the following 2 criteria:

1. the NTC tail-warping degree is lower than a preset range C, namely no tail-warping or slight tail-warping;

2. preferably selecting the probe with the smallest Ct value; (wherein, for low concentration plasmid template amplification, a Δct value <0.5 is considered no difference).

In practice, it is necessary to redesign primers or probes which are not normally amplified or which have abnormal amplified signals, and then perform the above-described test. The problems of single primer amplification, dimer peak (melting curve temperature is lower than target characteristic peak) existing in a low-concentration template, or primer probe amplification and slight tail tilting of NTC can be solved by system optimization, but the problems can increase difficulty in establishing a subsequent multiplex amplification system, so that the subsequent multiplex amplification system can be further tested, but the method is not preferable.

The primers and probes are screened, and no primers and probes are available except a few candidate target areas, and most candidate target areas screened through sequence alignment at least retain one primer probe combination. The primers and probes selected are as follows, wherein some primers or probes exist in more than one combination.

The sequences of the primers are shown in the following table, and the sequence numbers are SEQ ID NOs 1 to 44:

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For example, the sequence SEQ ID NO. 3 is the same sequence shared by Mycoplasma microorganisms Mycoplasma arginini, mycoplasma ORale, mycoplasma salivarium.

For example, SEQ ID NO. 8 is a sequence common to Mycoplasma microorganisms Mycoplasma synoviae, mycoplasma hyorhinis, mycoplasma fermentans.

For example, SEQ ID NO. 11 shows the same sequence as that shared by Mycoplasma microorganisms Mycoplasma salivarium, mycoplasma arginini.

For example, SEQ ID NO. 12 shows the same sequence as that shared by Mycoplasma microorganisms Mycoplasma gallisepticum, mycoplasma pneumoniae.

For example, SEQ ID NO. 13 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma hyorhinis, mycoplasma ORale, mycoplasma synoviae, mycoplasma salivarium.

For example, SEQ ID NO. 19 shows the same sequence as that shared by Mycoplasma microorganisms Mycoplasma arginini, mycoplasma salivarium.

For example, the sequence SEQ ID NO. 21 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma hyorhinis, mycoplasma fermentans.

For example, the sequence SEQ ID NO. 22 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma gallisepticum, mycoplasma pneumoniae.

For example, the sequence SEQ ID NO. 23 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma synoviae, mycoplasma hyorhinis, mycoplasma fermentans.

For example, the sequence SEQ ID NO. 26 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma hyorhinis, mycoplasma fermentans.

For example, the sequence SEQ ID NO. 31 is the same sequence as that common to Mycoplasma organisms Mycoplasma hyorhinis, mycoplasma ORale, mycoplasma synoviae, mycoplasma salivarium.

For example, SEQ ID NO. 33 shows the same sequence as that of Mycoplasma microorganisms Mycoplasma gallisepticum, mycoplasma pneumoniae.

For example, the sequence SEQ ID NO. 35 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma salivarium, mycoplasma arginini.

For example, sequence SEQ ID NO. 37 is a sequence common to Mycoplasma microorganism Mycoplasma arginini, mycoplasma orale, mycoplasma salivarium.

For example, SEQ ID NO. 38 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma gallisepticum, mycoplasma pneumoniae.

For example, sequence SEQ ID NO. 42 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma arginini, mycoplasma salivarium.

The remaining sequences include, for example, consensus sequences of other Mycoplasma microorganisms than Mycoplasma hyorhinis, mycoplasma arginini, mycoplasma gallisepticum, mycoplasma pneumoniae, etc.

The sequences of the probes are shown in the following table, with SEQ ID NOS: 45 to 66:

for example, SEQ ID NO. 45 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma gallisepticum, mycoplasma pneumoniae.

For example, SEQ ID NO. 49 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma salivarium, mycoplasma arginini.

For example, the sequence SEQ ID NO. 52 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma synoviae, mycoplasma hyorhinis, mycoplasma fermentans.

For example, sequence SEQ ID NO. 54 is a sequence common to Mycoplasma microorganisms Mycoplasma gallisepticum, mycoplasma pneumoniae.

For example, sequence SEQ ID NO. 58 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma hyorhinis, mycoplasma fermentans.

For example, the sequence SEQ ID NO. 60 is the same sequence as that common to Mycoplasma microorganisms Acholeplasma laidiawii, mycoplasma arginini.

For example, sequence SEQ ID NO. 62 is the same sequence as that common to Mycoplasma microorganisms Mycoplasma arginini, mycoplasma salivarium.

For example, the sequence SEQ ID NO. 66 is the same sequence as that common to Mycoplasma organisms Mycoplasma hyorhinis, mycoplasma ORale, mycoplasma synoviae, mycoplasma salivarium.

Specifically, the construction of the single amplification system in step S103 includes: step S1031 to step S1034.

Step S1031, optimizing primer concentration of each single amplification system;

step S1032, optimizing the annealing temperature of each single amplification system;

step S1033, optimizing the probe concentration of each single amplification system based on the optimized primer concentration and the annealing temperature;

step S1034, optimizing an amplification system of each single amplification system based on the primer concentration, the annealing temperature and the probe concentration obtained by the optimization.

Wherein in step S1034, the optimization of the amplification system of the single amplification system comprises at least one of:

selection of an amplification enzyme;

selecting a buffer solution;

the amount of dNTPs used;

mg ion concentration.

Specifically, primer concentration optimization and annealing temperature optimization were performed using the SYBR Green dye method.

Specifically, the primer concentration is optimized, the primer concentration gradient is set to 100nM/150nM/200nM, or 90nM/120nM/150nM, and the single primer pair is amplified (including the melting curve stage), and the amplification reaction can be referred to in step S102. The amplification template used plasmids with both a high and a low concentration gradient, or only a low concentration of plasmid was selected (100-200 copies/. Mu.L recommended).

Primer concentrations were screened according to the following 3 criteria:

1. the non-target peak of the melting curve is lower than a preset range D, and the Ct value of the amplification curve of the target is lower than a preset threshold E, namely the non-target peak of the melting curve is low or not;

2. the Ct value of the amplification curve of the target is lower than a preset threshold E, namely, primer concentration with smaller Ct value of the target is preferred;

3. after the conditions that the non-target peak of the melting curve is lower than the preset range D and the Ct value of the amplification curve of the target is lower than the preset threshold E are met, the primer concentration lower than other concentrations is preferably selected, namely, after the first two principles are met, the primer concentration is preferably lower.

The priorities of the above criteria are arranged in order from high to low according to items 1 to 3. As a result of the optimization, the concentration of the different primer pairs, although not uniform, is substantially between 100-150 nM.

Specifically, the annealing temperature was optimized and an annealing temperature gradient of 56/58/60/62 ℃ was set.

The annealing temperature was screened according to the following 3 standard criteria:

1. the non-target peak of the melting curve is lower than a preset range F, namely the non-target peak of the melting curve is lower or not, and the Ct value of the amplification curve of the target is lower than a preset threshold G, namely the Ct value of the amplification curve of the target is smaller (the amplification of a low-concentration plasmid template, delta Ct value is less than 0.5 and is considered to be no difference);

2. the difference value between the annealing temperatures of all the primers is lower than a preset difference value H, namely the annealing temperatures of all the primer pairs are as consistent as possible;

3. after the conditions that the non-target peak of the melting curve is lower than a preset range F, the Ct value of the amplification curve of the target is lower than a preset threshold G, and the difference value between the annealing temperatures of the primers is lower than a preset difference value H are met, the annealing temperature lower than other temperatures is preferably selected, namely, after the first two standards are preferably met, the lower annealing temperature is preferably selected.

As a result of the optimization, the annealing temperature is unified to about 60 ℃.

Specifically, the probe concentration is optimized (a pair of primers, a probe). And adding a matched probe into an amplification system by using the optimized primer concentration and annealing temperature, removing SYBR Green dye, and carrying out single-system amplification. The probe concentration gradient was set to 90nM/120nM/150nM, or 60nM/80nM/100nM. Typically, the probe concentration is no higher than the primer concentration.

Probe concentrations were screened according to the following criteria:

1. the Ct value of the amplification curve is lower than a preset threshold I, namely, the concentration of the probe is selected to have a small amplification Ct value (the amplification of a low-concentration plasmid template, the delta Ct value of <0.5 is considered to be no difference);

2. when the Ct values are the same, a probe concentration lower than the other concentrations is preferably selected, i.e., when the Ct values are the same, a lower probe concentration is selected.

In practice, the different probe concentrations are substantially uniform between 90 and 120 nM.

Specifically, optimization of an amplification system, including selection of amplification enzymes and buffers, selection of dNTP usage, adjustment of Mg ion concentration, and the like, can also directly screen commercial enzyme mix without adjusting specific components of the enzyme mix. 2-3 singleton system amplifications were performed using optimized primer probe concentration, annealing temperature, plasmid templates using both high and low concentration gradients, wherein the high concentration template used 1000 copies/. Mu.L and the low concentration template used 100 copies/. Mu.L. It is generally believed that the Ct value is smaller and that amplification systems with S-type amplification curves are better. Experimental tests several manufacturers have screened multiple amplification reagents such as gold, TAKARA, novalac, etc., and finally gold amplification reagent perfectStart II probe qPCR superMix UDG was preferred. According to the amplification reagent instructions, a recommended amplification procedure is used, wherein the annealing temperature uses an optimized temperature.

Specifically, the combining and combining the single amplification systems of at least two mycoplasma microorganisms corresponding to different species in step S104, and constructing a multiplex amplification system includes:

step S1041, combining preliminary single amplification systems by taking groups as units to obtain multiple amplification systems of each group;

step S1042, detecting, screening and optimizing the multiple amplification systems of each group by adopting the reaction conditions of the single amplification systems in the groups.

single amplification systems with repeated detection functions are excluded.

Specifically, a plurality of singleton systems are grouped according to the compatibility of the primer probes, and preliminary singleton system combination is performed by taking the group as a unit: the primers and probes which can form dimers are divided into different groups; the singleton systems that can share primers or probes are grouped as far as possible. In this example, multiple primer analyzer in on-line analysis software "Thermo Scientific Web Tools" was used to analyze whether dimers and interactions were present. Then, the preliminary grouping is simplified, 10 key strains in the table 1 are used as detection ranges, only one detection system of each strain is reserved, and the detection systems of the other targets are candidate for standby. Since more than one single system can be detected for each of the 10 key strains, there is a grouping of multiple different combinations of monomer systems.

And testing different monomer system combinations (groups) by using the optimized primer and probe concentration of the monomer system, the amplification reagent and the annealing temperature, and initially establishing a multiple detection system. The optimal test results are: the NTC (water template) of the multiplex detection system does not turn off the tail and the results have universal applicability to different instruments (qPCR instrument from 2-3 manufacturers tested). In the testing process of this embodiment, the preliminarily screened multiple system has slight tail-tilting of the NTC, or the NTC is not tail-tilting on one fluorescent quantitative PCR instrument, but the NTC is tail-tilting on another fluorescent quantitative PCR instrument. The tail-warping of the NTC can be further reduced or eliminated by optimizing an amplification system, such as primer probe concentration, amplification reagent components and amplification reaction conditions. The NTC tail, which cannot be eliminated, can be eliminated by limiting the threshold line and the amplification cycle number, but the sensitivity of the multiplex detection system is reduced.

Through several sets of experiments, the final preferred multiplex amplification system included the following:

a first multiplex amplification system.

The first sequence combination of primers of the first multiplex amplification system comprises:

The second sequence combination of probes of the first multiplex amplification system comprises:

(c3) SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 62 and SEQ ID NO. 66.

A second multiplex amplification system.

The first sequence combination of primers of the second multiplex amplification system comprises:

the second sequence combination of probes of the second multiplex amplification system comprises:

(c7) SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 60 and SEQ ID NO. 66.

In practical operation, the available primer probe combination modes are not only the above two, and the above two are selected, so that the detection range is mainly considered: the first multiplex amplification system and the second multiplex amplification system were able to detect all strains in table 1, with sequence alignments predicting hundreds of detectable mycoplasma, spiroplasma, and cholesterol-free pathogens.

Specifically, the first multiplex amplification system and the second multiplex amplification system are further optimized, and the method is similar to the single-strand system optimization (see step S103), except that the optimization of the multiplex system is performed in two steps:

1. The NTC is optimized so that it does not warp. The test results were that the primer and probe concentrations, especially the probe concentrations, were more pronounced to mitigate the NTC tail-flick effect.

2. The genomic DNA of the strain of interest (see Table 1) was used as template and the concentration gradients tested the lowest limit of detection for the different templates. The strain with lower detection sensitivity is detected, the concentration of the corresponding primer probe is properly increased, the primer probe with high detection sensitivity is correspondingly properly reduced, the total concentration of the probe is kept at a lower level, and the possibility of occurrence of tail warping of the NTC is reduced.

For each single detection system contained in the multiplex system, at least a corresponding one of the genomic DNA is used for the test. Finally, by optimization, the overall sensitivity of the first multiplex amplification system and the second multiplex amplification system (detection of 10 key strains) is close, but the second multiplex amplification system still has slight tailing, so the first multiplex amplification system is preferred.

step S105, setting internal reference detection;

Specifically, in order to monitor the abnormality of the amplification reaction, false negative results are avoided. It is necessary to add additional reference detection in a multiplex detection system. The internal reference may be selected to amplify any sequence different from mycoplasma, spiroplasma, and non-cholesteric sequences, such as the human-derived actin sequence. Selecting an internal reference target according to the target sequence of the primer probe in the multiplex amplification system, and designing an internal reference detection system compatible with the multiplex amplification system. Detection of internal controls uses VIC fluorescent-labeled probes to distinguish from the targets.

The design of the internal reference detection primer probe can refer to step S102, which is different in that:

the design of the primer probe of the internal reference mainly considers the compatibility with the multiple amplification system, can not generate dimer with the target detection system in the multiple amplification system, and has the Tm value as consistent as possible with the primer probe in the multiple amplification system.

The preliminary optimization of the internal reference system can refer to step S103, which differs in that:

the internal reference system only optimizes the primer probe concentration, and the amplification reagent and the amplification conditions are consistent with those of the multiplex amplification system.

After the internal participation multiplex amplification system is combined, the NTC can not tilt at the FAM fluorescent channel (target) and the VIC fluorescent channel (internal reference), and if the requirements are not met, the concentration of the internal reference primer probe needs to be further adjusted or the internal reference needs to be replaced again. Finally, the primer probe concentrations of the internal reference are all 100nM.

The primer sequences for internal reference detection are shown in the following table SEQ ID NO. 67 or SEQ ID NO. 68:

sequence numbering	Sequence(s)
		SEQ ID NO:67	5’-CCACCGCAAATGCTTCTAGG-3’
SEQ ID NO:68	5’-GCGCAAGTTAGGTTTTGTCAAG-3’

The probe sequence for internal reference detection adopts the sequence shown in the following table SEQ ID NO: 69:

sequence numbering	Sequence(s)
		SEQ ID NO:69	5’-FAM-CTATGACTTAGTTGCGTTACA-MGB-3’

In the first embodiment of the present invention, a multiplex detection system is established according to the steps S101 to S105. The primer probe of the combination 1 is used for target detection, the primer probe is used for internal reference detection, the concentration of each primer is in the range of 50-180nM, and the concentration of the probe is in the range of 50-120 nM. The amplified components are shown in Table 3 below and the reaction procedure is shown in Table 4 below. The threshold line for target detection (FAM channel) may be set to 0.12 and the threshold line for reference detection (VIC channel) may be set to 0.05.

Component (A)	1 reaction (mu L/rxn)
		perfectStart II probe qPCR superMix UDG	5
Primer probe combination	3.6
		ROX	0.4
Reference plasmid	1.0
		Template to be measured	10.0
Totals to	20.0

TABLE 3 multiplex amplification System Components

Table 4 multiplex system amplification procedure: AB 7500 fluorescent quantitative PCR instrument

It is possible for the person skilled in the art to substitute, delete, or add one or several nucleotides to the sequences shown in SEQ ID NOS 1 to 69.

Specifically, one skilled in the art can make base modifications to the primer sequences shown in SEQ ID NO. 1 to 44, make substitutions, deletions, or additions of one or more nucleotides to the first 10-15 nucleotides of the 5 'end, make modifications such as truncation to the 3' end sequence that do not alter the amplified product sequence or the altered product sequence has a similarity of greater than 90% to the original sequence and is identical in specificity. Such modifications are alternatives readily apparent to those skilled in the art and do not require the inventive effort and remain within the scope of the invention. The modified substitution of the internal reference detection primer sequence shown in SEQ ID NO. 67 or SEQ ID NO. 68 is the same as this.

Specifically, one skilled in the art can base modify the probe sequences shown in SEQ ID NOS.44 to 66, including forms in which the position of the amplified product can be shifted back and forth, the fluorescent signal and the quenching group can be changed, the probe length can be increased or decreased, the 5' -end of the probe can be modified or the linker sequence can be added, and the like. Such modifications are alternatives readily apparent to those skilled in the art and do not require the inventive effort and remain within the scope of the invention. The modified substitution of the reference detection probe sequence shown in SEQ ID NO. 69 is the same.

Example two

The primer probe combination for identifying mycoplasma provided by the embodiment II of the invention comprises the following primers: a first sequence combination; the first sequence combination includes: at least one consensus sequence;

the consensus sequence satisfies at least one of the following conditions:

the length of the consensus sequence is more than or equal to 30bp;

no more than 3 discrete degenerate bases of the consensus sequence;

Mycoplasma hyorhinis；

Mycoplasma arginini；

Mycoplasma gallisepticum；

Mycoplasma pneumoniae；

Acholeplasma laidiawii；

Mycoplasma fermentans；

Spiroplasma citri；

Mycoplasma synoviae；

Mycoplasma orale；

Mycoplasma salivarium。

in some alternative embodiments of the invention, the primer probe sequences of the single-amplification system are shown in the following table.

Microorganism name	Mycoplasma gallisepticum,Mycoplasma pneumoniae
		Primer sequence 1	SEQ ID NO:22
Primer sequence 2	SEQ ID NO:38
		Probe sequence	SEQ ID NO:54

Wherein the sequences SEQ ID NO. 22, SEQ ID NO. 38 and SEQ ID NO. 54 are the consensus sequences of Mycoplasma microorganisms Mycoplasma gallisepticum and Mycoplasma pneumoniae. The single amplification system corresponds to mycoplasma microorganisms Mycoplasma gallisepticum and Mycoplasma pneumoniae.

Microorganism name	Mycoplasma arginini,Mycoplasma salivarium
		Primer sequence 1	SEQ ID NO:19
Primer sequence 2	SEQ ID NO:42
		Probe sequence	SEQ ID NO:62

Wherein the sequences SEQ ID NO. 19, SEQ ID NO. 42 and SEQ ID NO. 62 are the consensus sequences of Mycoplasma microorganisms Mycoplasma arginini and Mycoplasma salivarium. The single amplification system corresponds to mycoplasma microorganisms Mycoplasma arginini and Mycoplasma salivarium.

Wherein the sequences SEQ ID NO. 13, SEQ ID NO. 31 and SEQ ID NO. 66 are the consensus sequences of Mycoplasma microorganism Mycoplasma hyorhinis, mycoplasma ORale, mycoplasma synoviae and Mycoplasma salivarium. The single amplification system corresponds to Mycoplasma microorganism Mycoplasma hyorhinis, mycoplasma orale, mycoplasma synoviae, mycoplasma salivarium.

Microorganism name	Mycoplasma arginini,Mycoplasma salivarium
		Primer sequence 1	SEQ ID NO:11
Primer sequence 2	SEQ ID NO:35
		Probe sequence	SEQ ID NO:49

Wherein the sequences SEQ ID NO. 11, SEQ ID NO. 35 and SEQ ID NO. 49 are the consensus sequences of Mycoplasma microorganisms Mycoplasma arginini and Mycoplasma salivarium. The single amplification system corresponds to mycoplasma microorganisms Mycoplasma arginini and Mycoplasma salivarium.

Microorganism name	Mycoplasma gallisepticum,Mycoplasma pneumoniae
		Primer sequence 1	SEQ ID NO:12
Primer sequence 2	SEQ ID NO:33
		Probe sequence	SEQ ID NO:45

Wherein the sequences SEQ ID NO. 12, SEQ ID NO. 33 and SEQ ID NO. 45 are the consensus sequences of mycoplasma microorganisms Mycoplasma gallisepticum and Mycoplasma pneumoniae. The single amplification system corresponds to mycoplasma microorganism Mycoplasma gallisepticum, mycoplasma pneumoniae.

Mycoplasma hyorhinis；

Mycoplasma arginini；

Mycoplasma gallisepticum；

Mycoplasma pneumoniae；

Acholeplasma laidiawii；

Mycoplasma fermentans；

Spiroplasma citri；

Mycoplasma synoviae；

Mycoplasma orale；

Mycoplasma salivarium。

in some alternative embodiments of the invention, the primer probe sequences of the multiplex amplification system are shown in the following table.

Wherein, 1 to 7 singles represent 7 singles of amplification systems, and reference represents reference detection. The single amplification system of items 1 to 7 as a whole constitutes a complete multiplex amplification system 1 which can detect common mycoplasma causing cell contamination, which should be detected by the European pharmacopoeia and Japanese pharmacopoeia regulations.

Wherein, 1 to 7 singles represent 7 singles of amplification systems, and reference represents reference detection. The single amplification system of items 1 to 7 as a whole constitutes a complete multiplex amplification system 2 which can detect common mycoplasma causing cell contamination, which should be detected by the European pharmacopoeia and Japanese pharmacopoeia regulations.

Wherein, 1 to 7 singles represent 7 singles of amplification systems, and reference represents reference detection. The single amplification system of items 1 to 6 as a whole constitutes a complete multiplex amplification system 3 which can detect common mycoplasma causing cell contamination, which should be detected by the European pharmacopoeia and Japanese pharmacopoeia regulations.

Furthermore, the primer probe combination for identifying mycoplasma,

the consensus sequence of the primer comprises:

Alternatively, the consensus sequence of the primer comprises:

(a2) SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 44, SEQ ID NO. 43, SEQ ID NO. 42, SEQ ID NO. 44, or a sequence which is deleted by a substitution or a sequence of the nucleotide sequence of the SEQ ID;

the consensus sequence of the probe comprises:

Alternatively, the consensus sequence of the probe comprises:

Furthermore, the primer probe combination for identifying mycoplasma,

the first sequence combination of primers comprises:

alternatively, the first sequence combination of primers comprises:

The second sequence combination of probes comprises:

alternatively, the second sequence combination of probes comprises:

the first sequence combination of primers comprises:

alternatively, the first sequence combination of primers comprises:

The second sequence combination of probes comprises:

alternatively, the second sequence combination of probes comprises:

The specific technical principle of the primer probe combination for identifying mycoplasma provided in the second embodiment of the present invention refers to the primer probe combination of the multiplex amplification system described in the second embodiment of the present invention, and is not described herein.

Example III

The third embodiment of the invention also provides a multiplex amplification system for identifying mycoplasma, which adopts the primer probe combination of the second embodiment of the invention.

the primer for internal reference detection comprises the following sequences:

(d1) SEQ ID NO. 67 or SEQ ID NO. 68;

The probes for reference detection include the following sequences:

(d3) The sequence shown in SEQ ID NO. 69;

the primer concentrations included: 100 to 150nM;

the annealing temperature includes: 55 to 65 ℃; preferably 58 to 62 ℃;

probe concentration includes; 50 to 100nM;

amplification enzymes include, but are not limited to: uracil-DNA glycosylase (UDG enzyme), DNA polymerase; wherein the DNA polymerase comprises: taq enzyme, high fidelity enzyme, or hot start enzyme;

buffers include, but are not limited to: tris (hydroxymethyl) aminomethane hydrochloride buffer (Tris-HCl buffer, pH 8.0), potassium chloride, magnesium sulfate, bovine Serum Albumin (BSA), glycerol, etc.;

the concentration of Mg ions includes: 3.2mM magnesium ion.

The specific technical principle of the multiplex amplification system provided in the third embodiment of the present invention refers to the multiplex amplification system in the first embodiment of the present invention, and is not described herein.

The embodiment of the invention also provides a kit for identifying mycoplasma, which adopts the primer probe combination in the second embodiment of the invention.

The embodiment of the invention also provides another kit for identifying mycoplasma, which adopts the multiplex amplification system in the third embodiment of the invention.

The embodiment of the invention also provides a kit for identifying mycoplasma, which adopts a multiplex amplification system;

the multiplex amplification system is obtained by adopting the construction method of the amplification system for identifying mycoplasma in the embodiment of the invention.

Performance verification for embodiments of the invention

Performance verification mainly includes three aspects of sensitivity, specificity and durability.

Sensitivity verification 10 key strains listed in table 1 were detected. Of these 7 were purchased quantitative strains (as shown in Table 5 below), and the minimum detection limit of 10CFU/mL was tested for Mycoplasma detection rate from nucleic acid extraction. 24 samples were taken for each strain and then qPCR assays were performed, requiring more than 90% of the samples to be detected.

The specific implementation scheme is as follows: based on quantitative information from purchased strains, 10-fold gradient dilutions were made to 10CFU/mL using nuclease-free water, and 200. Mu.L samples were taken for extraction. The nucleic acid extraction was performed using a commercial nucleic acid extraction kit, and in this example, a Tiangen nucleic acid extraction kit was used. Extraction was performed according to the instructions and finally, 50. Mu.L of nuclease-free water was used for elution and 10. Mu.L of nucleic acid eluate was used for qPCR detection. qPCR amplification System referring to Table 3, run procedure referring to Table 4, the apparatus used an AB 7500 fluorescent quantitative PCR apparatus.

The test results are shown in Table 5 below and in FIGS. 2 to 8 of the specification. The other 3 argininium, mycoplasma salivarius and spiroplasma citruses were tested using synthetic plasmids. The specific implementation scheme is as follows: the synthesized plasmid is quantified by a Qubit fluorometer, then diluted to 20copies/mL by ten-fold gradient with nuclease-free water, and directly taken to 10 mu L for qPCR detection. Sequence alignment showed that the target sequence contained 4copies in the Mycoplasma arginini genome, 7copies in the Mycoplasma arginini genome, and 2copies in the Mycoplasma citri, thus 20copies/mL of plasmid, with a theoretical minimum detection limit of no more than 10CFU/mL. qPCR amplification System referring to Table 3, run procedure referring to Table 4, the apparatus used an AB 7500 fluorescent quantitative PCR apparatus. The test results are shown in Table 6 below and in FIGS. 9 to 11 of the specification. Wherein reference numeral X1 denotes mycoplasma, and X2 denotes internal control.

TABLE 5 minimum detection limit detection results of seven strains after extraction

TABLE 6 minimum detection limit for the direct amplification detection of synthetic plasmids from three strains

The specificity verification includes cross test and interference test.

The crossover test is to detect the presence of amplification signals in other bacterial and mammalian DNA proximal to mycoplasma. Results of crossover test: ten near-border bacterial and fungal DNA amplifications (see table 7 below), without crossover (figure 12 of the specification); four mammalian DNA amplifications (see Table 8 below) without crossover (FIG. 13 of the specification). Wherein reference X3 denotes a target.

TABLE 7 10 near-field bacteria and fungi for specificity verification

Numbering device	DNA source	Dosage ng/test	Numbering device	Name of the name	Dosage ng/test
						1	Human leucocytes	10	3	Vero	10
2	CHO	10	4	sf9	10

TABLE 8 4 mammalian cells for specificity verification

The interference test is to evaluate the effect of the co-extraction of the interfering substance and mycoplasma on the amplification, and the deviation of average Ct value is generally < + -0.5, so that the interference has no effect on detection. The interfering substances were tested by selecting 5 components and media common in cell culture. Results of the interference test: five sample matrices had no effect on mycoplasma (. About.100 copies/mL) detection (see Table 9).

	Mycoplasma synoviae	PBS	Horse serum	Arginine (Arg)	DMEM	RPMI
							Extraction 1	30.10	29.82	30.06	29.94	29.94	29.87
Extraction 2	29.85	29.94	29.91	29.92	29.88	29.90
							Average value of	29.98	29.88	29.99	29.93	29.91	29.89
ΔCt	——	-0.09	0.01	-0.05	-0.07	-0.09
								Mycoplasma gallisepticum	PBS	Horse serum	Arginine (Arg)	DMEM	RPMI
Extraction 1	30.19	29.95	30.53	29.94	29.98	29.96
							Extraction 2	29.96	30.13	30.05	29.76	30.04	29.86
Average value of	30.08	30.04	30.29	29.85	30.01	29.91
							ΔCt	——	-0.04	0.21	-0.23	-0.07	-0.17

TABLE 9 influence of five media on Ct values detected after extraction of Mycoplasma synoviae and Mycoplasma gallisepticum

In terms of durability, three aspects of freeze-thawing stability, storage stability at 4 ℃ and batch-to-batch stability of the detection reagent were evaluated.

The test method of freeze thawing stability comprises the following steps: split charging the detection reagent, detecting mycoplasma plasmid after one part of freeze thawing for five times, and directly detecting the same mycoplasma plasmid in one part. The detection results are shown in Table 10, and the detection results are not affected by the five times of freezing and thawing of the detection reagent.

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Table 10 summary of freeze-thaw stability data for three batches of detection reagents

The test method for the storage stability at 4 ℃ is as follows: the detection reagent is packaged and stored at 4 ℃. Every 4 days, for 2 weeks. And a new sub-packaging reagent is taken for testing each time of detection, so that repeated use is avoided. The results are shown in Table 11, and the detection reagent is stable when stored at 4℃for 16 days.

Storing the reagent at 4 DEG C	Ct value 1	Ct value 2	Average value of	Delta Ct value (n-0 days)
					Day 0	26.91	26.77	26.84	——
For 4 days	27.22	27.14	27.18	0.34
					For 8 days	27.10	26.96	27.03	0.19
For 12 days	26.97	26.93	26.95	0.11
					For 16 days	27.02	26.98	27.00	0.16

Table 11 summary of storage stability data at 4 °

The testing method of the stability among batches comprises the following steps: different operators, on different dates, use different batches of detection reagents to carry out the sample detection of the lowest detection limit concentration of the genomic DNA or synthetic plasmid of different mycoplasma (without cholestasis and spiroplasma). The detection results are shown in Table 12, and the positive detection rates are all above 90%.

Table 12 summary of data for different lots of test minimum limit samples

* Wherein, the Mycoplasma arginini, mycoplasma salivarius and Mycoplasma citri are detected by using synthetic plasmids, and the rest of genome DNA is detected.

The sequence number of each process in the embodiments of the present application does not mean the sequence of the sequence, and the sequence of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the present application.

In addition, the term "and/or" herein is merely an association relationship describing an association object, and means that three relationships may exist, for example, a and/or B may mean: a exists alone, A and B exist together, and B exists alone. In addition, the character "/" herein generally indicates that the front and rear associated objects are an "or" relationship.

The foregoing embodiments have been provided for the purpose of illustrating the general principles of the present application in further detail, and are not to be construed as limiting the scope of the application, but are merely intended to cover any modifications, equivalents, improvements, etc. based on the teachings of the application.

Claims

1. A method of constructing an amplification system for identifying mycoplasma, comprising:

2. The method for constructing an amplification system for identifying mycoplasma according to claim 1, wherein the step of designing primers and probes comprises:

establishing a common sequence search region of mycoplasma microorganism;

searching the consensus sequence search region for a consensus sequence capable of covering at least one of Zhi Yuanti, spiroplasmataceae, and cholesterless families;

the consensus sequence is selected according to at least one of the following criteria:

The length of the consensus sequence is more than or equal to 30bp;

no more than 3 discrete degenerate bases of the consensus sequence;

3. The method of constructing an amplification system for the identification of mycoplasma as claimed in claim 2, wherein the consensus sequence is capable of covering at least one mycoplasma microorganism of the group consisting of:

Mycoplasma hyorhinis；

Mycoplasma arginini；

Mycoplasma gallisepticum；

Mycoplasma pneumoniae；

Acholeplasma laidiawii；

Mycoplasma fermentans；

Spiroplasma citri；

Mycoplasma synoviae；

Mycoplasma orale；

Mycoplasma salivarium。

4. the method for constructing an amplification system for mycoplasma identification according to claim 1, wherein the step of screening the primer and the probe comprises:

based on the primers obtained by the screening, the probes are screened.

5. The method for constructing an amplification system for identifying mycoplasma as claimed in claim 1, wherein the step of constructing a single amplification system comprises:

Optimizing the annealing temperature of each single amplification system;

selection of an amplification enzyme;

selecting a buffer solution;

the amount of dNTPs used;

mg ion concentration.

6. The method of claim 1, wherein the step of combining at least two of the single amplification systems corresponding to different mycoplasma microorganisms to construct a multiplex amplification system comprises:

single amplification systems with repeated detection functions are excluded.

7. The method for constructing an amplification system for identifying mycoplasma as claimed in claim 1, further comprising, after the step of constructing a multiplex amplification system:

setting internal reference detection;

8. The method for constructing an amplification system for mycoplasma identification according to claim 7,

the primer for internal reference detection comprises the following sequences:

(d1) SEQ ID NO. 67 or SEQ ID NO. 68;

the probes for reference detection include the following sequences:

(d3) The sequence shown in SEQ ID NO. 69;

9. The method for constructing an amplification system for identifying mycoplasma as claimed in any one of claim 1 to 8,

the primers of the single amplification system comprise the following consensus sequences:

the probe of the single amplification system comprises the following consensus sequences:

and the primers in the same single amplification system are matched with the probes so as to ensure that the amplification reaction with consistent specificity of the primer amplification products can be carried out.

10. The method for constructing an amplification system for mycoplasma identification according to claim 9,

or alternatively, the process may be performed,

11. The primer probe combination for identifying mycoplasma is characterized in that,

the primer comprises: a first sequence combination; the first sequence combination includes: at least one consensus sequence;

12. The primer probe combination for identifying mycoplasma as claimed in claim 11,

the consensus sequence is capable of covering at least one of Zhi Yuanti, spiroplasmaceae, and cholesteryl-free families;

the consensus sequence satisfies at least one of the following conditions:

the length of the consensus sequence is more than or equal to 30bp;

no more than 3 discrete degenerate bases of the consensus sequence;

13. The primer probe combination for identifying mycoplasma as claimed in claim 11,

the primer of the first sequence combination and the probe of the second sequence combination belong to a multiplex amplification system;

14. The primer probe combination for identifying mycoplasma according to claim 11, wherein said single amplification system corresponds to at least one mycoplasma microorganism of the following:

Mycoplasma hyorhinis；

Mycoplasma arginini；

Mycoplasma gallisepticum；

Mycoplasma pneumoniae；

Acholeplasma laidiawii；

Mycoplasma fermentans；

Spiroplasma citri；

Mycoplasma synoviae；

Mycoplasma orale；

Mycoplasma salivarium；

Mycoplasma hyorhinis；

Mycoplasma arginini；

Mycoplasma gallisepticum；

Mycoplasma pneumoniae；

Acholeplasma laidiawii；

Mycoplasma fermentans；

Spiroplasma citri；

Mycoplasma synoviae；

Mycoplasma orale；

Mycoplasma salivarium。

15. the primer probe combination for identifying mycoplasma according to any one of claim 11 to 14,

the consensus sequence of the primer comprises:

the consensus sequence of the probe comprises:

16. The primer probe combination for identifying mycoplasma as claimed in claim 15,

the first sequence combination of primers comprises:

The second sequence combination of probes comprises:

or alternatively, the process may be performed,

the first sequence combination of primers comprises:

the second sequence combination of probes comprises:

17. A multiplex amplification system for identifying mycoplasma, characterized in that it employs a primer probe combination according to any one of claims 11 to 16.

18. The multiplex amplification system for the identification of mycoplasma as claimed in claim 17, further comprising: detecting internal parameters;

the primer for internal reference detection comprises the following sequences:

(d1) SEQ ID NO. 67 or SEQ ID NO. 68;

the probes for reference detection include the following sequences:

(d3) The sequence shown in SEQ ID NO. 69;

19. The multiplex amplification system for the identification of mycoplasma according to claim 17, wherein the reaction conditions of the multiplex amplification system are as follows:

The primer concentrations included: 100 to 150nM;

the annealing temperature includes: 55 to 65 ℃;

probe concentration includes; 50 to 100nM;

the dNTP dosage comprises: 100. Mu.M dATP, 100. Mu.M dGTP, 100. Mu.M dTTP, 100. Mu.M dCTP, 10. Mu.M dUTP;

the concentration of Mg ions includes: 3.2mM magnesium ion.

20. Kit for the identification of mycoplasma, characterized in that it uses a primer probe combination according to any one of claims 11 to 16.

21. Kit for the identification of mycoplasma, characterized in that it employs a multiplex amplification system according to any one of claims 17 to 19.

22. A kit for identifying mycoplasma, characterized in that the kit employs a multiplex amplification system;

the multiplex amplification system is obtained by the construction method of the amplification system for identifying mycoplasma according to any one of claims 1 to 10.