Nucleic acid combination product, kit for detecting genital tract infection pathogens and detection method

Abstract

The invention relates to a nucleic acid combination product, a kit for detecting genital tract infection pathogens and a detection method. The nucleic acid combination product provided by the invention comprises a composition of a plurality of groups of primer pairs and detection probes, is wide in detection coverage and high in inclusiveness, can simultaneously detect a plurality of genital tract infection pathogens in a single-tube reaction system, and has relatively high sensitivity and specificity; the kit is a genital tract infection pathogen detection product with comprehensive detection targets at present, can quickly and accurately provide a basis for clinical diagnosis, and provides support for prevention, control and treatment of genital tract infection.

Description

Technical Field

[0001] The present application belongs to the technical field of pathogenic microorganism detection, and specifically relates to a nucleic acid combination product, a kit for detecting pathogens of reproductive tract infections, and a detection method. Background Art

[0002] Reproductive tract infections (RTIs) are a group of reproductive tract infections caused by a variety of pathogens, including bacteria, viruses, mycoplasmas, fungi, and Trichomonas. These infections are widely spread through sexual contact (accounting for over 90%), blood-borne transmission, and mother-to-child transmission, impacting reproductive health. Sexually transmitted diseases (STDs) are diseases that develop from sexually transmitted infections and are the most common type of RTI.

[0003] Faced with the severe situation of reproductive tract infections, traditional detection methods have significant limitations. The smear microscopy method has the disadvantages of being cumbersome, time-consuming, and having low sensitivity, making it difficult to meet the requirements of early, rapid, and accurate diagnosis. Although the culture method is the "gold standard," it takes several days to several weeks, and fastidious bacteria (such as chlamydia) or viruses are more difficult to culture. Serological testing is limited by the antibody window period and cannot distinguish between current infection and past infection. These defects have led to a high rate of missed diagnosis, seriously delaying treatment. With the development of molecular biology technology, nucleic acid testing has gradually become the core means of diagnosing reproductive tract infections with its advantages of high sensitivity, high specificity, and rapid detection.

[0004] Currently, a variety of test kits for reproductive tract infections are commercially available, but 90% offer single-test functionality, 10% offer dual or triple-test functionality, and very few manufacturers offer triple or higher testing. Furthermore, traditional reproductive tract testing typically involves collecting samples through swabs or secretions, an invasive procedure that can easily cause discomfort or even pain in patients. Some individuals, fearing pain, delay or refuse screening. Therefore, developing a product or technology that can detect multiple common reproductive tract pathogens with high sensitivity and specificity is crucial. Summary of the Invention

[0005] Based on this, one embodiment of the present application provides a nucleic acid combination product, a kit for detecting pathogens of reproductive tract infections, and a detection method.

[0006] On the one hand, the present application provides a nucleic acid combination product, which includes multiple sets of the following probe primer combinations:

[0007] (1) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 2, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 3, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 4;

[0008] (2) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 5 to SEQ ID NO: 6, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 7, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 8;

[0009] (3) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 9 to SEQ ID NO: 10, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 11, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 12;

[0010] (4) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 9 to SEQ ID NO: 10, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 13, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 14;

[0011] (5) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 15 to SEQ ID NO: 16, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 17, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 18;

[0012] (6) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 19 to SEQ ID NO: 20, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 21, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 22;

[0013] (7) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 23 to SEQ ID NO: 24, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 25, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 26;

[0014] (8) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 23 to SEQ ID NO: 24, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 27, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 28;

[0015] (9) A primer pair having a nucleotide sequence as shown in SEQ ID NO: 29 to SEQ ID NO: 30, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 31, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 32.

[0016] In some embodiments, the nucleic acid combination product includes primers and probes having nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 32.

[0017] In some embodiments, the 3' end of the PCO detection probe is labeled with a phosphate group; the two ends of the THO detection probe are respectively labeled with a fluorescent reporter group and a fluorescent quencher group;

[0018] Optionally, the 5' end of the THO detection probe is labeled with the fluorescent reporter group, and the 3' end is labeled with the fluorescent quencher group;

[0019] Optionally, the fluorescent reporter group includes one or more of FAM, VIC, ROX and CY5;

[0020] Optionally, the fluorescence quenching group includes one or more of BHQ1 and BHQ2.

[0021] In some embodiments, the nucleic acid combination product further includes an internal reference gene detection primer pair and / or an internal reference gene detection probe;

[0022] Optionally, the internal reference gene detection primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO.33 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.34;

[0023] Optionally, the nucleotide sequence of the internal reference gene detection probe is shown in SEQ ID NO.35.

[0024] Another aspect of the present application provides a kit for detecting pathogens of reproductive tract infections, characterized in that the kit comprises: the above-mentioned nucleic acid combination product.

[0025] In some embodiments, the kit further comprises: one or more of a nucleic acid releasing reagent, a nucleic acid extraction reagent, and a nucleic acid amplification reagent;

[0026] Optionally, the nucleic acid amplification reagent includes one or more of DNA polymerase, dNTPs, UNG enzyme, PCR buffer and Mg2+.

[0027] In some embodiments, the kit further comprises one or more of a positive control and a negative control.

[0028] In some embodiments, the positive control comprises one or more of a Mycoplasma genitalium control, a Mycoplasma hominis control, and a Trichomonas vaginalis control;

[0029] Or / and, the negative control product includes enzyme-free water.

[0030] Another aspect of the present application provides a method for detecting pathogens of reproductive tract infection, comprising:

[0031] Providing a nucleic acid sample to be tested, performing a PCR amplification reaction on the nucleic acid sample to be tested using the aforementioned nucleic acid combination product or the aforementioned kit, analyzing the resulting amplification product using a melting curve method, and determining whether the nucleic acid sample to be tested contains a pathogen of reproductive tract infection based on the obtained analysis results;

[0032] Optionally, the PCR amplification reaction includes an MPA multiplex fluorescence PCR amplification reaction.

[0033] In some embodiments, the source of the nucleic acid sample to be tested includes one or more of genital tract swabs, genital tract secretions, and urine.

[0034] The nucleic acid combination product provided in this application has a wide detection coverage and can simultaneously detect multiple reproductive tract infection pathogens in a single-tube reaction system with high sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0036] Figure 1 The amplification curve and melting curve characteristic spectrum results of Neisseria gonorrhoeae in the FAM channel in Example 2, where the left figure is the amplification curve, the horizontal axis is the cycle number, and the vertical axis Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the horizontal axis is the temperature, and the vertical axis -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity with respect to temperature, which is used to more clearly show the rate of change of the fluorescence signal with temperature. In the melting curve analysis, it can help determine the melting temperature (Tm value) of the product;

[0037] Figure 2The amplification curve and melting curve characteristic spectrum results of Mycoplasma genitalium in the FAM channel in Example 2 are shown. The left figure is the amplification curve, the horizontal axis is the cycle number, and the vertical axis Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the horizontal axis is the temperature, and the vertical axis -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity with respect to temperature;

[0038] Figure 3 The amplification curve and melting curve characteristic spectrum results of Chlamydia trachomatis in the FAM channel in Example 2, where the left figure is the amplification curve, the abscissa is the cycle number, and the ordinate Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the abscissa is the temperature, and the ordinate -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity with respect to temperature;

[0039] Figure 4 The amplification curve and melting curve characteristic spectrum results of the VIC channel Ureaplasma urealyticum in Example 2, wherein the left figure is the amplification curve, the abscissa is the cycle number, and the ordinate Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the abscissa is the temperature, and the ordinate -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity with respect to temperature;

[0040] Figure 5 The amplification curve and melting curve characteristic graphs of Mycoplasma hominis in the VIC channel in Example 2 are shown. The left figure is the amplification curve, with the abscissa representing the cycle number and the ordinate Rn representing the fluorescence signal intensity. The right figure is the melting curve, with the abscissa representing the temperature and the ordinate -d(Rn) / dT representing the negative first-order derivative of the fluorescence signal intensity with respect to temperature.

[0041] Figure 6 The amplification curve and melting curve characteristic spectrum results of the microscopic Ureaplasma in the VIC channel in Example 2, where the left figure is the amplification curve, the abscissa is the cycle number, and the ordinate Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the abscissa is the temperature, and the ordinate -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity with respect to temperature;

[0042] Figure 7 The amplification curve and melting curve characteristic spectrum results of herpes simplex virus type 1 in the ROX channel in Example 2, wherein the left figure is the amplification curve, the horizontal axis is the cycle number, and the vertical axis Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the horizontal axis is the temperature, and the vertical axis -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity with respect to temperature;

[0043] Figure 8 The amplification curve and melting curve characteristic spectrum results of herpes simplex virus type II in the ROX channel in Example 2, wherein the left figure is the amplification curve, the horizontal axis is the cycle number, and the vertical axis Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the horizontal axis is the temperature, and the vertical axis -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity with respect to temperature;

[0044] Figure 9 The amplification curve and melting curve characteristic spectrum results of Trichomonas vaginalis in the ROX channel in Example 2, where the left figure is the amplification curve, the horizontal axis is the cycle number, and the vertical axis Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the horizontal axis is the temperature, and the vertical axis -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity with respect to temperature;

[0045] Figure 10 The amplification curve and melting curve characteristic spectrum results of the six negative samples in Example 3 are shown in the figure on the left, where the amplification curve is the abscissa, the cycle number is the horizontal axis, and the vertical axis Rn refers to the fluorescence signal intensity; the melting curve is shown on the right, the horizontal axis is the temperature, and the vertical axis -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity with respect to temperature;

[0046] Figure 11 The amplification curve and melting curve characteristic graphs of the clinical urine sample in Application Example 4 are shown in the figure on the left, where the amplification curve is the cycle number on the horizontal axis and the fluorescence signal intensity is the vertical axis Rn; the melting curve is shown on the right, where the horizontal axis is the temperature and the vertical axis -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity with respect to temperature.

[0047] Figure 12 The amplification curve and melting curve characteristic graphs of the positive quality control in Application Example 4 are shown in the figure on the left, where the amplification curve is the cycle number on the horizontal axis and the fluorescence signal intensity is the vertical axis Rn; the melting curve is shown on the right, where the horizontal axis is the temperature and the vertical axis -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity with respect to the temperature. DETAILED DESCRIPTION

[0048] Below in conjunction with embodiment and example, the application is described in further detail.Should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description hereinafter, a large amount of specific details are given in order to provide a more complete understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0050] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0051] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0052] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0053] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0054] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0055] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0056] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0057] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0058] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0059] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0060] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0061] As shown in the background art, among the pathogens of reproductive tract infections, the pathogenicity and harmfulness of Neisseria gonorrhoeae, Chlamydia trachomatis, Herpes Simplex Virus, and Trichomonas vaginalis are particularly prominent. Neisseria gonorrhoeae mainly invades the human cervix, urethra, rectum, eyes, or throat, causing local inflammation and suppurative lesions, and causing gonorrhea, a common sexually transmitted disease. Globally, there are 125,000 cases of infertility caused by gonorrhea each year, the risk of ectopic pregnancy increases by 7 times, and the incidence of chronic pelvic pain exceeds 20%. Chlamydia trachomatis is known for its "invisible transmission". About 70% of female and 50% of male infected people are asymptomatic. Among untreated female infected people, 40% develop pelvic inflammatory disease. In addition, fallopian tube damage significantly increases the possibility of infertility. Genital herpes is a sexually transmitted infection caused by herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Symptoms primarily include painful blisters and ulcers in the genital area, causing localized pain and itching, with a cyclical course. Globally, approximately 491 million people aged 15-49 are infected with HSV-2. Studies have shown that HSV-2 infection increases the risk of HIV transmission by 2-3 times. Neonatal herpes mortality from mother-to-child transmission can reach 60%. Trichomonas vaginalis, the most common non-viral sexually transmitted parasite worldwide, accounts for approximately 276 million new cases annually, accounting for 15%-20% of all sexually transmitted infections. Symptoms primarily include increased vaginal discharge, vulvar itching, and burning. Trichomonas vaginitis can damage the genital mucosal barrier, and infection during pregnancy can increase the risk of premature birth and premature rupture of the placenta.

[0062] In addition to the pathogens mentioned above, common reproductive tract pathogens include Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, and Mycoplasma genitalium. Ureaplasma urealyticum and Ureaplasma parvum are carried in sexually active populations at rates as high as 40%-80%, and are closely associated with adverse pregnancy outcomes. Studies have shown that infection with Ureaplasma urealyticum in pregnant women can increase the risk of chorioamnionitis by 1.5 times. As the second most common pathogen of non-gonococcal urethritis (NGU) (accounting for 30%-40%), Mycoplasma genitalium poses a significant threat. Approximately 1%-3% of adults worldwide carry this pathogen, and the incidence of cervicitis in infected women exceeds 50%.

[0063] Based on this, the present application provides a nucleic acid combination product on one hand, which includes multiple sets of the following probe primer combinations:

[0064] (1) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 2, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 3, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 4;

[0065] (2) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 5 to SEQ ID NO: 6, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 7, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 8;

[0066] (3) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 9 to SEQ ID NO: 10, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 11, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 12;

[0067] (4) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 9 to SEQ ID NO: 10, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 13, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 14;

[0068] (5) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 15 to SEQ ID NO: 16, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 17, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 18;

[0069] (6) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 19 to SEQ ID NO: 20, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 21, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 22;

[0070] (7) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 23 to SEQ ID NO: 24, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 25, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 26;

[0071] (8) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 23 to SEQ ID NO: 24, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 27, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 28;

[0072] (9) A primer pair having a nucleotide sequence as shown in SEQ ID NO: 29 to SEQ ID NO: 30, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 31, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 32.

[0073] The nucleic acid combination product provided in this application has a wide detection coverage and can simultaneously detect multiple reproductive tract infection pathogens in a single-tube reaction system with high sensitivity and specificity.

[0074] In some embodiments, the nucleic acid combination product includes primers and probes having nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 32.

[0075] It can be understood that the present application uses multiplex fluorescent probe PCR technology (Multiplex Probe Amplification, MPA) to develop a detection kit for 9 reproductive tract infection pathogens using melting curve analysis as a typing method, covering a comprehensive range of pathogens and rapidly detecting pathogen types in a single tube. The principle of MPA technology is: each target is provided with a pair of primers, a fluorescently labeled probe (THO), and a hybridization probe (PCO) that is reverse complementary to the THO probe and introduces mismatched bases. After PCR amplification, during the process of increasing the melting curve analysis temperature (e.g., 25°C to 75°C), the THO-PCO hybrid double strand undergoes a process of fluorescence from presence to absence, thus forming a melting curve with a unique Tm (Melting Temperature). Depending on the degree of mismatch introduced into the THO-PCO hybrid double strand and the different mismatched bases, each fluorescent channel can accommodate the melting curve peaks of multiple targets, and the difference between adjacent melting curve peaks is at least 4°C. When the target gene is not present in the reaction system, such as a negative quality control, the THO probe will not be cleaved by the Taq enzyme during the amplification stage, and will form a melting peak with the PCO during the melting curve stage. When the target gene is present in the reaction system, the THO probe is cleaved by the Taq enzyme during the amplification phase. The higher the target concentration, the more the THO probe is consumed, up to the point of complete consumption. During melting curve analysis, the melting peak heights of THO and PCO decrease or disappear. By comparing the melting curves with those of a negative control sample in which THO is not consumed, the target type present in the sample can be determined, enabling qualitative and typing detection of different pathogens in the sample.

[0076] In some embodiments, the 3' end of the PCO detection probe is labeled with a phosphate group; and the two ends of the THO detection probe are respectively labeled with a fluorescent reporter group and a fluorescent quencher group.

[0077] In some embodiments, the 5' end of the THO detection probe is labeled with the fluorescent reporter group, and the 3' end is labeled with the fluorescent quencher group.

[0078] In some embodiments, the fluorescent reporter groups include one or more of FAM, VIC, ROX, and CY5. It is understood that these groups have different absorbance values and can select different channels, so they will not interfere with each other.

[0079] In some embodiments, the fluorescence quenching group includes one or more of BHQ1 and BHQ2.

[0080] In some embodiments, the present application utilizes MPA multiplex fluorescent probe PCR technology to simultaneously detect nine common reproductive tract infection pathogens and perform typing via melting curve analysis. The nine common reproductive tract pathogens include: Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma genitalium, Mycoplasma Hominis, Herpes simplex virus 1, Herpes simplex virus 2, and Trichomonas vaginalis.

[0081] In some embodiments, the fluorescent reporter group of the THO detection probes for Neisseria gonorrhoeae, Chlamydia trachomatis, and Mycoplasma genitalium is FAM, and the fluorescent quencher group is BHQ1; the fluorescent reporter group of the THO detection probes for Ureaplasma urealyticum, Ureaplasma parvum, and Mycoplasma hominis is VIC, and the fluorescent quencher group is BHQ1; the fluorescent reporter group of the THO detection probes for herpes simplex virus type 1, herpes simplex virus type 2, and Trichomonas vaginalis is ROX, and the fluorescent quencher group is BHQ2.

[0082] In some embodiments, the nucleic acid combination product further includes an internal reference gene detection primer pair and / or an internal reference gene detection probe.

[0083] In some embodiments, the above-mentioned internal reference gene detection primer pair includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.33 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.34.

[0084] In some embodiments, the nucleotide sequence of the internal reference gene detection probe is shown in SEQ ID NO. 35. The specific sequence is shown in Table 1 below.

[0085] Table 1

[0086]

[0087] Another embodiment of the present application provides a kit for detecting pathogens of reproductive tract infections, wherein the kit includes the above-mentioned nucleic acid combination product.

[0088] In some embodiments, the above-mentioned kit further comprises: one or more of a nucleic acid releasing reagent, a nucleic acid extraction reagent and a nucleic acid amplification reagent.

[0089] In some embodiments, the nucleic acid amplification reagent includes DNA polymerase, dNTPs, UNG enzyme, PCR buffer and Mg 2+ One or more of .

[0090] In some embodiments, the above-mentioned kit further includes one or more of a positive quality control product and a negative quality control product.

[0091] In some embodiments, the positive control comprises one or more of a Mycoplasma genitalium control, a Mycoplasma hominis control, and a Trichomonas vaginalis control.

[0092] In some embodiments, the negative control comprises enzyme-free water.

[0093] Another embodiment of the present application further provides a method for detecting pathogens of reproductive tract infection, comprising:

[0094] A nucleic acid sample to be tested is provided, and a PCR amplification reaction is performed on the nucleic acid sample to be tested using the above-mentioned nucleic acid combination product or the above-mentioned kit. The obtained amplification product is analyzed using a melting curve method, and the presence of reproductive tract infection pathogens in the nucleic acid sample to be tested is determined based on the obtained analysis results.

[0095] In some embodiments, the PCR amplification reaction comprises an MPA multiplex fluorescence PCR amplification reaction.

[0096] In some embodiments, the source of the nucleic acid sample to be tested includes one or more of genital tract swabs, genital tract secretions, and urine.

[0097] In some embodiments, the step of analyzing the reaction results includes respectively obtaining the Ct value and melting curve characteristic spectrum of the THO detection probe amplification, and determining the result according to the Ct value and the melting curve characteristic spectrum.

[0098] The above-mentioned multiplex fluorescence PCR detection kit and detection method for multiple nucleic acid detection of reproductive tract infection pathogens can detect 9 types of reproductive tract infection pathogens by typing in a single tube with high sensitivity and high specificity. Through MPA multiple fluorescence technology, multiple sexually transmitted pathogens can be detected simultaneously in a single tube, which helps to improve the diagnosis level of reproductive tract infections, promote early diagnosis, reduce the infection rate, and provide a basis for better guiding the formulation of clinical treatment plans.

[0099] In addition, since pathogens are highly polymorphic, they can also be used for research in fields such as sociology (eg, bacterial species distribution, etc.). The detection method includes both detection methods involving disease diagnosis and detection methods not involving disease diagnosis.

[0100] (1) This application is based on multiplex fluorescent probe PCR technology (MPA) and can achieve simultaneous detection of nine common reproductive tract pathogens, which improves the detection capacity of currently available test kits, can quickly and accurately lock single or multiple pathogen infection types, and reduce the risk of cross-contamination and the occurrence of false positive results; in addition, this application is compatible with all conventional fluorescent quantitative PCR instruments, and the operation process is simple and fast, saving time and cost, and providing strong support for the accurate diagnosis, treatment and prevention of reproductive tract infections.

[0101] (2) The detection sensitivity of this application is strong, and the sensitivity of each target can reach 1 copy per reaction, which is of great significance for asymptomatic infected persons and low-load samples, and helps in the early identification, treatment and prevention of reproductive tract infections, reducing the risk of missed diagnosis.

[0102] (3) The detection specificity of this application is strong. Through experimental verification, this kit effectively avoids cross-reactions with pathogens close to the target, common non-target pathogens of the reproductive tract, or the human genome, thereby improving the accuracy and efficiency of clinical diagnosis and facilitating accurate medication and targeted treatment by doctors.

[0103] (4) This application is compatible with different types of samples. Users can choose non-invasive urine sampling, which greatly reduces discomfort during use, enhances patients' willingness to test for reproductive tract pathogens, and is beneficial for reproductive tract infection screening.

[0104] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0105] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0106] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0107] Example 1

[0108] 1. The primer-probe combination used in the present application for detecting nine pathogens of reproductive tract infection includes a THO detection probe, a PCO detection probe, and a primer pair, the sequences of which are shown in Table 1.

[0109] The nucleic acid combination product for nine reproductive tract infection pathogens also includes the following reagents:

[0110] ②Negative control product: enzyme-free water;

[0111] ② Positive quality control: plasmid containing Mycoplasma genitalium, Mycoplasma hominis, Trichomonas vaginalis and internal standard fragment;

[0112] ③ Enzyme mixture (Nanjing Novozymes, QN222).

[0113] 2. Reagent parameters are shown in Table 2:

[0114] Table 2

[0115] Final primer concentration Final probe concentration Enzyme mixture concentration 200nM 100nM 1×

[0116] 3. The reagent formula is shown in Table 3:

[0117] Table 3

[0118] Main components Addition volume (μL) / test Enzyme mixture 10 Primer probe working solution 2

[0119] Enzyme-free water 3 total 15

[0120] (1) Take out the kit, thaw it at room temperature, mix all the components, centrifuge quickly, and place on ice until ready for use.

[0121] (2) Calculate the number of tests required for the test (n = number of samples + positive quality control + negative quality control + 1) and prepare the reaction solution according to the formula in Table 3.

[0122] (3) Mix the prepared PCR reaction solution, centrifuge quickly, dispense 15 μL / well into the corresponding PCR tubes, and transfer to the sample processing area for use.

[0123] 4. Sample processing (sample processing area)

[0124] The test sample in this embodiment is urine, and a nucleic acid extraction and purification kit produced by Guangzhou Baochuang Biotechnology Co., Ltd. is used to extract nucleic acid from the urine sample.

[0125] (1) Urine enrichment. 5 mL of urine was placed in a 15 mL conical centrifuge tube and centrifuged at 12,000 rpm for 5 min. The supernatant was discarded and 200 μL of the precipitate was retained. 20 μL of resuspension buffer was added, vortexed for 25 seconds, and incubated at 95°C for 20 min.

[0126] (2) Add the extraction reagent. First, remove the pre-packaged reagent kit and invert it several times to resuspend the magnetic beads. Gently shake or briefly spin the plate to concentrate the reagent and magnetic beads at the bottom of the plate. Carefully remove the plate sealing film. Then, add 20 μL of proteinase K solution to the 1st or 7th column of the pre-packaged reagent, and then add 200 μL of sample.

[0127] (3) Extraction on the machine. Place the deep-well plate in the nucleic acid extraction instrument and select the preset "Germ-Fast" process according to the extraction reagent instructions for automated extraction.

[0128] 5. Sample addition (sample processing area)

[0129] Add 5 μL each of negative control, sample extract, and positive control to the reaction solution in sequence, cover the reaction tube tightly, mark it, centrifuge for 30 seconds, and transfer it to the PCR amplification area.

[0130] 6. PCR amplification (amplification region)

[0131] The PCR reaction program was set according to Table 4 below, and the amplification volume was set to 20 μL.

[0132] Table 4

[0133]

[0134] 7. Result judgment criteria:

[0135] (1) Determination of baseline: The baseline can be automatically adjusted by the instrument or adjusted according to the actual amplified background signal;

[0136] (2) Determination of threshold: The threshold is generally set at the inflection point of the sample exponential amplification period and is higher than the baseline background. The setting can be adjusted according to the actual amplification situation. The baseline and threshold should be determined separately for each channel;

[0137] (3) Use the quality control products in the kit for quality control

[0138] Positive quality control: FAM, VIC, ROX and CY5 all have amplification curves with Ct≤35. At the same time, the melting curves corresponding to FAM, VIC and ROX are typed as Mycoplasma genitalium, Mycoplasma hominis and Trichomonas vaginalis.

[0139] Negative quality control: no amplification curve in each channel or Ct value > 40.

[0140] The sample test results can only be interpreted after the negative quality control and positive quality control are judged to be qualified.

[0141] (4) Interpretation method:

[0142] First, analyze whether the internal control is amplified in the CY5 channel. If no amplification is observed or the Ct value is >35, this indicates that the sample concentration is too low or that interfering substances are inhibiting the reaction, and the experiment needs to be re-prepared. If CY5 amplifies and the Ct value is ≤35, the result is valid and subsequent analysis can proceed. If the sample amplifies with a Ct value ≤40 in the FAM channel, or (and) the HEX / VIC channel, or (and) the ROX channel, this indicates that the FAM, (and) HEX / VIC, or (and) ROX target is positive. The target type needs to be determined by melting curve characteristic patterns. Specific criteria and methods are shown in Table 5.

[0143] Table 5

[0144]

[0145] Example 2 Sensitivity Verification

[0146] This example tested the sensitivity of the primer and probe combinations described above using ddPCR-based quality control samples for Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma genitalium, Mycoplasma hominis, herpes simplex virus type 1, herpes simplex virus type 2, and Trichomonas vaginalis. Nine quality control nucleic acid samples were diluted to 200 copies / mL using TE buffer (i.e., 1 copy / reaction) and tested in 20 replicates using the kit provided herein. Sterile purified water was used as the NTC.

[0147] The sensitivity test results of the above 9 samples are as follows: Figures 1 to 9 As shown in the figure, the minimum detection limit of each target can reach 1 copy / reaction, and the detection rate of 20 replicates is 100%, indicating that this reagent has good sensitivity.

[0148] Example 3 Specificity Verification

[0149] Cross-reaction tests were conducted on common pathogenic nucleic acids of reproductive tract infections outside the detection range of the above-mentioned kits, human genomic nucleic acids, and nucleic acids of pathogens close to the targets of the above-mentioned kits, including 6 negative quality control products: human genomic DNA quantitative standard material, Hela cell line, Candida albicans, Escherichia coli, Mycoplasma pneumoniae, and Neisseria meningitidis.

[0150] The results are as follows Figure 10 As shown, the kit prepared in the present application tested the above 6 samples according to the detection method of Example 1, and there was no amplification curve and no cross reaction, indicating that the reagent has good specificity.

[0151] Example 4 Clinical Sample Testing

[0152] In this example, 10 urine clinical samples were collected, and the nucleic acid samples were extracted and tested using the above-mentioned kit in this application to evaluate the ability of the kit to detect actual samples. Figure 11 As shown, there are 8 cases with amplification curves among the above samples, and the test results are positive. Figure 11 The results of (clinical samples) and Figure 12 (positive control) results were consistent.

[0153] Table 6

[0154] Sample No. FAM HEX / VIC ROX CY5 Pathogen identification Y1 NoCt 38.98 NoCt 25.19 Ureaplasma parvum Y2 NoCt 27.86 NoCt 26.03 Ureaplasma parvum Y3 NoCt NoCt 28.29 22.08 HSV-1 Y4 NoCt NoCt NoCt 18.26 Negative Y5 NoCt NoCt NoCt 17.05 Negative Y6 35.92 NoCt NoCt 32.93 Chlamydia trachomatis Y7 NoCt 30.42 NoCt 25.63 Mycoplasma hominis Y8 NoCt 31.57 NoCt 27.11 Ureaplasma parvum Y9 NoCt 31.47 NoCt 25.70 Ureaplasma urealyticum Y10 NoCt 31.99 NoCt 28.74 Ureaplasma parvum

[0155] According to the judgment criteria of melting peak and Tm value in Example 1, the type of pathogen in clinical positive samples can be obtained, and the results are shown in Table 6. The results of this example show that the primer probe combination and kit provided by this application can normally reflect the detection results of clinical samples.

[0156] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A nucleic acid combination product, characterized in that: The nucleic acid combination product includes multiple groups of the following probe primer combinations: (1) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 2, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 3, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 4; (2) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 5 to SEQ ID NO: 6, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 7, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 8; (3) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 9 to SEQ ID NO: 10, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 11, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 12; (4) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 9 to SEQ ID NO: 10, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 13, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 14; (5) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 15 to SEQ ID NO: 16, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 17, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 18; (6) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 19 to SEQ ID NO: 20, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 21, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 22; (7) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 23 to SEQ ID NO: 24, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 25, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 26; (8) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 23 to SEQ ID NO: 24, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 27, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 28; (9) A primer pair having a nucleotide sequence as shown in SEQ ID NO: 29 to SEQ ID NO: 30, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 31, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO:

32.

2. The nucleic acid combination product according to claim 1, characterized in that The nucleic acid combination product includes primers and probes with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO:

32.

3. The nucleic acid combination product according to claim 1, characterized in that The 3' end of the PCO detection probe is labeled with a phosphate group; the two ends of the THO detection probe are respectively labeled with a fluorescent reporter group and a fluorescent quencher group; Optionally, the 5' end of the THO detection probe is labeled with the fluorescent reporter group, and the 3' end is labeled with the fluorescent quencher group; Optionally, the fluorescent reporter group includes one or more of FAM, VIC, ROX and CY5; Optionally, the fluorescence quenching group includes one or more of BHQ1 and BHQ2.

4. The nucleic acid combination product according to claim 1, characterized in that The nucleic acid combination product further comprises an internal reference gene detection primer pair and / or an internal reference gene detection probe; Optionally, the internal reference gene detection primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO.33 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.34; Optionally, the nucleotide sequence of the internal reference gene detection probe is shown in SEQ ID NO.

35.

5. A kit for detecting pathogens of reproductive tract infection, characterized in that: The kit comprises: the nucleic acid combination product according to any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that The kit further comprises: one or more of a nucleic acid releasing reagent, a nucleic acid extracting reagent, and a nucleic acid amplification reagent; Optionally, the nucleic acid amplification reagent includes DNA polymerase, dNTPs, UNG enzyme, PCR buffer and Mg 2+ One or more of .

7. The kit according to claim 5 or 6, characterized in that The kit also includes one or more of a positive quality control product and a negative quality control product.

8. The kit according to claim 7, characterized in that The positive quality control products include one or more of Mycoplasma genitalium quality control products, Mycoplasma hominis quality control products and Trichomonas vaginalis quality control products; Or / and, the negative control product includes enzyme-free water.

9. A method for detecting pathogens of reproductive tract infection, characterized in that: include: Providing a nucleic acid sample to be tested, performing a PCR amplification reaction on the nucleic acid sample to be tested using the nucleic acid combination product according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 8, analyzing the obtained amplification product using a melting curve method, and determining whether the nucleic acid sample to be tested contains a pathogen of reproductive tract infection based on the obtained analysis results; Optionally, the PCR amplification reaction includes an MPA multiplex fluorescence PCR amplification reaction.

10. The method for detecting pathogens of reproductive tract infection according to claim 9, characterized in that: The source of the nucleic acid sample to be tested includes one or more of genital tract swabs, genital tract secretions and urine.

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