JP2015532114A - Methods and compositions for detection of nucleic acid targets from biological samples and body fluids - Google Patents

Abstract

The present invention is directed to a rapid sample preparation method, which involves crude crude biological sample lysates (eg, human urine, saliva, blood, urethra and cervical swabs, and biological material). Amplification directly from other samples) allows detection of highly sensitive and specific target nucleic acids (eg, human genomic DNA, human pathogen genomic DNA, human non-pathogenic genomic DNA). The gist of the present invention is to describe components of a biological sample that can be rapidly released from human and pathogenic cells, and that can be adapted for subsequent nucleic acid amplification methods. As an example of application, the present invention also discloses protocols and primer sequences for isothermal nucleic acid amplification (recombinase polymerase amplification (RPA), loop mediated isothermal amplification (LAMP)). The above enables highly specific and sensitive diagnosis of Homo sapiens, Chlamydia trachomatis and Mycoplasma genitalium genomic material from crude biological sample lysates and / or purified total DNA . This case amplification can be used in conjunction with immunochromatographic product detection using lateral flow strips to enable rapid (less than 20 minutes) isothermal nucleic acid amplification of human urine samples for C. trachomatis and M. genitalium diagnostics To do. The above does not require special laboratory equipment or qualified personnel and is therefore well suited for application in a point-of-care environment. [Selection figure] None

Description

  The present invention is directed to compositions and methods for rapid pretreatment of biological samples, which pretreatment allows detection by nucleic acid amplification of target nucleic acids from subsequent biological samples and body fluids.

  Conventional diagnosis relies primarily on nucleic acid amplification technology (NAAT). The most commonly known method for amplification of specific DNA is PCR, which provides reasonable laboratory level sensitivity. New technologies that have recently emerged have evolved from isothermal amplification (eg, recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), helicase dependent amplification). These isothermal NAATs do not require thermocycling of the reaction, yet exhibit a very high level of sensitivity, resulting in an amplification product that can be detected from as few as 1-2 template copies. Isothermal reactions make the technology well adapted to point-of-care (POC) environments (eg, general practitioners, homes), bringing diagnostic tests to the patient in a simple and rapid manner, and further reducing time to results To do. In the field of sexually transmitted diseases, POC diagnosis also significantly reduces the spread of relevant pathogens, especially those that are asymptomatic, such as C. trachomatis and M. genitalium Enables private and non-invasive testing with the potential to

  Both M. genitalium and C. trachomatis infections are known as “silent” diseases because they often remain asymptomatic. Therefore, regular diagnostic screening of these sexually transmitted pathogens is very important. Traditionally, C. trachomatis infection has been diagnosed by tissue culture methods from urethra or cervical swabs. Since the culture only identifies viable C. trachomatis cells, the sensitivity of the diagnosis is affected by the freshness of the specimen, which depends on the time from collection to laboratory processing. Thus, in the 1980s, antigen and nucleic acid detection techniques have been developed with the aim of C. trachomatis diagnostics that require less cost, time, experience, and conservation of infectivity during transport. Furthermore, nucleic acid detection technology has been demonstrated to have a much higher level of sensitivity because it can detect pathogen DNA from non-viable cells or cell debris. Biological detection of M. genitalium is also performed exclusively by specific amplification of pathogen DNA by PCR. M. genitalium culture is extremely difficult and is not routinely performed. The serological detection method of M. genitalium has low sensitivity and specificity.

Although NAAT provided a significant opportunity for highly effective diagnosis, it has only been routinely used to date only at the laboratory level. NAAT is complex to implement and requires skilled personnel and expensive machinery. Therefore, NAAT diagnosis is concentrated in large hospitals and diagnostic centers. One of the major limitations of NAAT technology is that a pure DNA sample is required. The purity of the sample can significantly affect the performance of NAAT (especially PCR). Novel isothermal NAATs such as RPA, LAMP, HDA, etc. appear to be less sensitive to nucleic acid sample purity and can efficiently amplify DNA present in human urine samples, for example.
The present invention discloses a method and its compounds for pretreatment of biological samples, which pretreatment enables efficient release of genomic DNA from cellular material. The sample pretreatment method described is compatible with subsequent nucleic acid amplification procedures that allow detection of target DNA from crude sample lysates. The present invention allows the omission of DNA purification steps prior to NAAT analysis and thus has a significant impact on the complexity and speed of the diagnostic technique. The present invention significantly facilitates the implementation of highly sensitive and specific NAAT diagnosis in a POC environment.

Since the examples of the present invention are exclusively directed to human sexually transmitted pathogenetic diagnosis, an overview of Chlamydia trachomatis and Mycoplasma genitalium will be provided below.
C. trachomatis and M. genitalium are human sexually transmitted pathogens. They are both associated with male nongonococcal (nonspecific) urethritis and several inflammatory genital tract syndromes in women (eg cervicitis and pelvic inflammatory disease). Inflammatory diseases caused by acute naïve infections of C. trachomatis and M. genitalium are one of the leading causes of female infertility worldwide.
The prevalence of M. genitalium ranges from 1-4% for men and 1-6% for women. Epidemic data in reported high-risk populations (eg, within a sexually transmitted disease (STD) testing center) reaches 38%. The prevalence of C. trachomatis among young adults with active sexual activity is 5-10%, depending on age, race, etc. C. trachomatis infection is almost always more prevalent among women and has been increasing worldwide over the past decades.

M. genitalium is a small (0.2-0.3 μm) polymorphic bacterium that lacks the cell wall, which makes it resistant to common antibiotics that target the cell wall (eg penicillin). M. genitalium has a unique adherent organelle in a bottle-like form. The cell organ allows bacteria to adhere to various substances and cells, including human epithelial cells. Adhesion is a major mechanism of M. genitalium pathogenesis, which requires at least seven adhesins, including the major adhesin MgPa (encoded by the MgPB gene).
C. trachomatis is a Gram-negative obligate intracellular pathogen with a unique biphasic growth cycle that exists as two developmental types (EB (or elementary body) and RB (or network-like structure)) . EB is the smaller (0.2 μm) metabolically inactive infectious extracellular form of the organism and RB is the larger (0.8 μm) metabolically active intracellular form. Chlamydia infection requires attachment of EBs to host cells and subsequent internalization into membrane-bound vesicles. Inclusions differentiate into RB, which undergoes 8-12 cell divisions utilizing host cell ATP and metabolites. RB differentiates and matures into infectious EBs and is released by host cell lysis. C. trachomatis strains are serologically classified into 15 serotypes based on antigenic changes of major outer membrane proteins. AC serotypes are ocular pathogens that cause ocular trachoma. Serotypes D-K and L1-L2 are sexually transmitted pathogens that infect columnar epithelial cells of the genital tract.

Adaptive immunity against C. trachomatis involves an INF-γ-mediated host cell response that deprives Chlamydia RB of tryptophan and ultimately interferes with the ability of trachomatis to grow and replicate. The C. trachomatis genital serotype carries several eubacterial tryptophan biosynthetic genes (TRPA and TRPB encoding the alpha and beta subunits of tryptophan synthase that catalyze the conversion of indole to tryptophan). Thus, the C. trachomatis genital serotype possesses the ability to utilize exogenous indoles secreted by the normal bacterial flora of the genital tract, which allows the trachomatis to overcome IFN-γ-mediated growth limitations and Support infection establishment.
M. genitalium has a small AT-rich (68%) 0.58 Mb genome (encoding 485 genes). Despite its small size, 4% of the genome consists of repetitive elements (MgPa repeats) that show homology with the MGPB gene. C. trachomatis also carries a small genome of approximately 1 Mb chromosome and a 7.5 kb latent plasmid. Almost all C. trachomatis strains carry 4 to 10 plasmid copies per chromosome. Several C. trachomatis isolates that do not contain plasmids have also been reported, but their virulence is significantly reduced compared to plasmid-bearing strains. The Chlamydia plasmid sequence is highly conserved (less than 1% mutation) and contains eight major coding sequences (CDS) along with an origin of replication formed by four 22 bp tandem repeats. Computer analysis confirmed that the plasmid-encoded protein has a function in replication.

The present invention discloses methods and compounds for pretreatment of biological samples that allow efficient release of genomic DNA from cellular material. A major advantage of the disclosed sample pretreatment method is its suitability for subsequent nucleic acid amplification procedures that allow detection of target DNA from crude sample lysates. Thus, the present invention allows the omission of DNA purification steps prior to NAAT analysis and thus has a significant impact on the complexity and speed of the diagnostic technique.
The present invention discloses a cytolytic compound, which releases genomic material rapidly (within 5 minutes at RT ° C.) and efficiently from mammalian cells, their pathogens and symbiotic microorganisms, bacteria and fungal cultures, etc. . The sample pretreatment buffer consists of a membrane active (cell penetrating) peptide, a mild detergent, or a combination of the two.
Membrane active peptides have antibacterial and antimicrobial effects that act destructively on bacterial membranes. They are also known as transmembrane drugs that can deliver various cargo molecules to mammalian cells (eg oligonucleotides, siRNA, plasmids, peptides). The present invention is directed to a novel use of cell penetrating peptides for diagnostic purposes. At higher (μM-mM) concentrations, the cell penetrating peptide disrupts the cell membrane, allowing the release of genomic DNA that can be used as a target in subsequent nucleic acid amplification reactions. Cell membrane disrupting peptides have no or very little inhibitory effect on nucleic acid amplification, even at high concentrations, and can therefore be used efficiently as genomic material release promoters.

Surfactants are very good solubilizers, but they tend to denature proteins by destroying the original three-dimensional structure. Mild ionic or non-ionic surfactants (eg Triton X-100, Triton X-114, NP-40, CHAPS, octyl-β-glucoside, octyl-β-thio) at low concentrations (eg 0.1-1%) Certain combinations of glucopyranoside) efficiently destroy the cell wall and release the genomic material encapsulated within the cell. These mild surfactants do not significantly impair the nucleic acid amplification procedure and can induce or facilitate the release of a sufficient amount of the target nucleic acid. In order to efficiently lyse the cells within 5 minutes of incubation at RT ° C., the detergent composition and concentration are set.
The ability of membrane active peptide and / or detergent mediated sample pretreatment to convert biological samples into materials that can be successfully used for nucleic acid amplification is a major aim of the present invention, said ability to be rough This was confirmed by establishing detection of genomic DNA of Chlamydia trachomatis, Mycoplasma genitalium and human (Homo sapiens) by human urine lysate.

For this purpose, a highly specific and sensitive diagnostic method for the detection of C. trachomatis and M. genitalium in human samples is based on isothermal nucleic acid amplification (RPA, LAMP) and immunochromatography using lateral flow strips. Developed with product detection. For both pathogens we used a dual target system where simultaneous detection of two different genomic targets was performed. The above reduces the probability of a false negative diagnostic result when a deletion or mutation is introduced into the genomic DNA region of the pathogen used as an amplification target. All target regions were selected on the basis of their high homology among various pathogen strains and lack of identity with similar species.
For the detection of C. trachomatis, we used a genomic sequence derived from a well-established diagnostic target, the coding sequence 2 (CDS2) of a multicopy latent plasmid. For the second target, we selected the β subunit of the tryptophan synthase gene TRPB. For M. genitalium detection, we used the genomic sequence of the gene encoding MgPa dominant adhesin (MGPB), a major component of multi-repeat throughout its genome. For the second target, we used the 16S rRNA gene (also present in multiple copies within the M. genitalium genome). However, the M. genitalium 16S rRNA gene is highly conserved among various mycoplasma species (eg 98% identity with M. pneumonia, M. gallisepticum) 91% identity). Therefore, regions containing multiple mutations were selected for isothermal amplification and additional specificity tests were performed for this particular target.

For each target, an optimal primer pair combination was established that allows the highest level of sensitivity in the assay. The optimized RPA reaction allowed the amplification of a well detectable detectable stable product that yielded a minimum of 20-50 target sequence copies. Optimized LAMP reaction with loop primers allowed product amplification that yielded a minimum of 5-10 target sequence copies. Each diagnostic target uses 50,000 copies (0.16 ng) of H. sapiens genomic DNA and, in the case of M. genitalium 16S rRNA target, also using 100,000 copies of M. pneumonia genomic DNA Were tested. The sensitivity and specificity of isothermal amplification was verified by total DNA extracted from human urine samples.
The main objective of the present invention was to develop a diagnostic assay applicable under point-of-care conditions. Therefore, we have incorporated immunochromatographic amplification product detection into this diagnostic assay. For this purpose, the forward primer sequence was labeled 5 'with biotin and the reverse primer was labeled with fluorescein amidite (FAM). A double-labeled product was produced during the amplification reaction, which was detected within minutes using a lateral flow strip. Incorporation of immunochromatographic product detection required additional primer optimization. Primers that obtain a signal that the template-independent lateral flow strip can detect were excluded from the selection.
The diagnostic method by RPA and LAMP isothermal amplification has also been shown to be suitable for simultaneous detection of multiple targets. Both assays were optimized for the H. sapiens GAPDH gene target used as a positive control for diagnostic testing of human samples. PCR and isothermal amplification (RPA / LAMP / HDA) protocols were adjusted for optimal sensitivity and high specificity of the diagnostic test.

The method for detecting a nucleic acid target from a crude biological sample and body fluid comprises the following steps:
a) lysis of cells and the release of nucleic acid targets in biological samples and body fluids (eg, tissues, urine, saliva, blood, stool, hair, etc. and derivatives thereof, but not limited to these illustrative lists) Sample pretreatment, where a lytic peptide is used to release the nucleic acid target in the biological sample;
b) Amplification of nucleic acids, including nucleic acids such as DNA, RNA and their derivatives (but not limited to the above list), amplification is initiated by the presence of the target, eg PCR (polymerase chain reaction), HCR (hybridization chain reaction) , Amplification methods such as RCA (rolling circle amplification), RPA (recombinase polymerase amplification), LAMP (loop-mediated isothermal amplification), HDA (helicase-dependent amplification) and the like (but not limited to the above list of examples) Where the sequence based on one or more specific targets is amplified or the sample solution obtained during step (1) is directly subjected to further amplification procedures;
c) Detection of amplification products, including the use of qualitative or quantitative detection methods, such as sandwich assay, ELISA (enzyme linked immunosorbent assay), LF (lateral flow) immunochromatographic assay, wavelength varying (Visible spectrum, chemiluminescent, fluorescent, phosphorescent) dyes, denrimere, etc., or detection molecules and ligands to which the relevant moieties are bound, including optical devices, appropriate wavelength launchers or readers or combinations thereof In some cases, qualitative and quantitative detection is performed on a crude sample solution.

The pretreatment method is specifically designed to detect the following nucleic acid targets:
-Chlamydia trachomatis (using specific target regions provided in Table 1 or using specific primers and / or labeled derivatives thereof provided in Tables 2, 3); and-Mycoplasma genii Thallium (use specific target regions provided in Table 1 or use specific primers and / or labeled derivatives thereof provided in Tables 2 and 3).
The present invention using the human genome GAPDH target
-As internal verification and platform evaluation technology,
-Used for detection as an internal verification and platform evaluation technique with specific primers and / or labeled derivatives thereof provided in Tables 2 and 3.
This pretreatment method relates to molecular diagnosis of Chlamydia trachomatis, where the TRPB gene is used as a molecular diagnostic target.
Example

Rapid diagnosis of the presence of Chlamydia trachomatis in urine samples This protocol describes methods and components thereof for the highly sensitive diagnosis of Chlamydia trachomatis in human urine samples. The entire procedure, including sample pretreatment, target isothermal amplification and product detection, takes less than 20 minutes and requires 10 minutes incubation at 37 ° C. The described method detects two C. trachomatis targets, a TRPB sequence in the genomic region and a CDS2 sequence in the latent plasmid region (Table 1).

両C.トラコマチス標的は、同じ標識を有する高度に特異的で鋭敏なプライマーを用いて増幅される（フォワードプライマーはビオチンで標識され、リバースプライマーはFAMで標識される）。したがって、C.トラコマチスに特異的な生成物は、ラテラルフローストリップ上での免疫クロマトグラフィー検出時に区別されない。2つのC.トラコマチス領域の検出は、該標的領域の一方が変異又は欠失する場合に陽性検査結果を担保するために用いられる。該反応はまたH.サピエンスGAPDH遺伝子を標的とするプライマーを含み、前記プライマーはDIG及びFAM標識生成物を生じる。この生成物は、ラテラルフローストリップ上で分離したレーンとして認識され、全手順（細胞のゲノム物質の放出、増幅及び検出）のための陽性コントロールとして供される。記載の方法の分析感度は、一検査につき50個のC.トラコマチス細胞及び50個のH.サピエンス細胞である。これは、起床時尿（first void urine）の尿1mL当たり10,000細胞以上の病源体濃度のC.トラコマチスの検出を可能にする。
患者の尿サンプルを等体積のサンプル前処理緩衝液（0.2％トリトンX-114、150mM NaCl、50mMトリス（pH 7.0）を含む）と混合し、RT℃で5分インキュベートする。10μLの前記処理サンプルをRPA反応に用いる。前記RPA反応は以下の成分を含む：C.トラコマチスPL-CDS2 の5'ビオチン標識FW3プライマー（最終濃度0.4μM）、C.トラコマチスPL-CDS2 の5' FAM標識RV1プライマー（最終濃度0.4μM）、C.トラコマチスTRPBの 5'ビオチン標識FW2プライマー（最終濃度0.4μM）、C.トラコマチスTRPBの5'FAM標識RV3プライマー（最終濃度0.4μM）、H.サピエンスGAPDHの5'DIG標識FW3プライマー（最終濃度0.4μM）、H.サピエンスGAPDHの5'FAM標識RV2プライマー（最終濃度0.4μM）（プライマー配列については表2を参照されたい）、14mM酢酸マグネシウム、TwistDX RPA酵素ペレット及び 29.5μLの再水和緩衝液。反応物を37℃で10分間インキュベートする。稀釈緩衝液を用い生成物を1：10比に稀釈し、ビオチン-FAM及びDIG-FAM標識分子を検出するラテラルフローストリップで分析する。 Table 1: Genomic regions of Chlamydia trachomatis, Mycoplasma genitalium and Homo sapiens used for detection by isothermal amplification

Both C. trachomatis targets are amplified using highly specific and sensitive primers with the same label (the forward primer is labeled with biotin and the reverse primer is labeled with FAM). Thus, products specific for C. trachomatis are not distinguished upon immunochromatographic detection on lateral flow strips. Detection of two C. trachomatis regions is used to ensure a positive test result if one of the target regions is mutated or deleted. The reaction also includes a primer targeting the H. sapiens GAPDH gene, which yields DIG and FAM labeled products. This product is recognized as a separate lane on the lateral flow strip and serves as a positive control for the entire procedure (release, amplification and detection of cellular genomic material). The analytical sensitivity of the described method is 50 C. trachomatis cells and 50 H. sapiens cells per test. This allows detection of C. trachomatis with a pathogen concentration of 10,000 cells or more per mL of urine in first void urine.
Patient urine samples are mixed with an equal volume of sample pretreatment buffer (containing 0.2% Triton X-114, 150 mM NaCl, 50 mM Tris, pH 7.0) and incubated at RT ° C. for 5 minutes. 10 μL of the treated sample is used for RPA reaction. The RPA reaction includes the following components: C. trachomatis PL-CDS2 5 ′ biotin labeled FW3 primer (final concentration 0.4 μM), C. trachomatis PL-CDS2 5 ′ FAM labeled RV1 primer (final concentration 0.4 μM), C. trachomatis TRPB 5 'biotin labeled FW2 primer (final concentration 0.4μM), C. trachomatis TRPB 5'FAM labeled RV3 primer (final concentration 0.4μM), H. sapiens GAPDH 5'DIG labeled FW3 primer (final concentration) 0.4 μM), H. sapiens GAPDH 5 ′ FAM labeled RV2 primer (final concentration 0.4 μM) (see Table 2 for primer sequences), 14 mM magnesium acetate, TwistDX RPA enzyme pellet and 29.5 μL rehydration buffer liquid. Incubate the reaction at 37 ° C. for 10 minutes. The product is diluted 1:10 using dilution buffer and analyzed on a lateral flow strip that detects biotin-FAM and DIG-FAM labeled molecules.

検査が2本線（ビオチン-FAM及びDIG-FAM）を示す場合クラミジア検査は陽性であり、検査が1本線（DIG-FAM）を示す場合陰性である。いずれの線も存在しないか又はビオチン-FAM線のみが存在する場合、検査の結果は無効である。 Table 2: Specific primer sequences for the targets provided in Table 1 for recombinase polymerase amplification

Chlamydia test is positive if the test shows two lines (biotin-FAM and DIG-FAM), and negative if the test shows one line (DIG-FAM). If none of the lines are present or only the biotin-FAM line is present, the test result is invalid.

Rapid diagnosis of the presence of Mycoplasma genitalium in urine samples This protocol describes a method and components thereof for highly sensitive Mycoplasma genitalium diagnosis in human urine samples. The entire procedure, including sample pretreatment, target isothermal amplification and product detection, takes less than 20 minutes and requires 10 minutes incubation at 37 ° C. The described method detects two M. genitalium targets, MGPA and 16S rRNA sequences in the pathogen genome (Table 1). Both M. genitalium targets are amplified using highly specific and sensitive primers with the same label (the forward primer is labeled with biotin and the reverse primer is labeled with FAM). Thus, products specific for M. genitalium are not distinguished upon immunochromatographic detection on lateral flow strips.

Detection of two M. genitalium regions is used to ensure a positive test result if one of the target regions is mutated or deleted. The reaction also includes a primer targeting the H. sapiens GAPDH gene, which yields DIG and FAM labeled products. This product is recognized as a separate lane on the lateral flow strip and serves as a positive control for the whole procedure (release, amplification and detection of cellular genomic material). The analytical sensitivity of the described method is at least 50 M. genitalium cells and 50 H. sapiens cells per test. This allows the detection of M. genitalium with a pathogen concentration of 10,000 cells or more per mL of urine in waking urine.
A patient urine sample is mixed with an equal volume of sample pretreatment buffer (containing 0.2% NP-40, 150 mM NaCl, 50 mM Tris, pH 7.0) and incubated at RT ° C. for 5 minutes. 10 μL of the treated sample is used for RPA reaction. The RPA reaction includes the following components: M. genitalium MGPA 5 ′ biotin labeled FW4 primer (final concentration 0.4 μM), M. genitalium MGPA 5 ′ FAM labeled RV4 primer (final concentration 0.4 μM), M. Genitalium 16S rRNA 5 'biotin labeled FW1 primer (final concentration 0.4μM), M. genitalium 16S rRNA 5' FAM labeled RV1 primer (final concentration 0.4μM), H. sapiens GAPDH 5 'DIG labeled FW3 primer (final concentration 0.4μM) Final concentration 0.4 μM), H. sapiens GAPDH 5 ′ FAM labeled RV2 primer (final concentration 0.4 μM) (see Table 2 for primer sequences), 14 mM magnesium acetate, TwistDX RPA enzyme pellet and 29.5 L re-water Sum buffer. Incubate the reaction at 37 ° C. for 10 minutes. The product is diluted 1:10 using dilution buffer and analyzed on a lateral flow strip that detects biotin-FAM and DIG-FAM labeled molecules.
The M. genitalium test is positive if the test shows two lines (biotin-FAM and DIG-FAM) and negative if the test shows one line (DIG-FAM). If none of the lines are present or only the biotin-FAM line is present, the test result is invalid.

The method uses highly sensitive loop-mediated isothermal amplification (LAMP) for specific detection of C. trachomatis DNA. The analytical sensitivity of the described method is at least 5 C. trachomatis cells per test. The LAMP reaction is prepared as follows: C. trachomatis PL-CDS2 set 4 primers F3 and B3 (each concentration 0.2 μM), C. trachomatis PL-CDS2 set 4 5 ′ per 25 μL reaction. Biotin-labeled FIP and 5 ′ FAM-labeled BIP primers (1.6 μM each), C. trachomatis PL-CDS2 set 4 5 ′ biotin-labeled LF and 5 ′ FAM-labeled LB loop primers (each 0.8 μM) (for primer sequences) See Table 3), 5.6 μM dNTP, 6 mM MgSO ₄ , 0.8 M betaine, 8 units of Bst polymerase, 2.5 μL of 10 × Bst polymerase buffer and 5 μL of total DNA extracted from patient samples. The reaction is incubated at 63 ° C. for 1 hour, diluted to a 1:10 ratio with dilution buffer, and analyzed on a lateral flow strip that detects biotin-FAM labeled molecules.
In a similar reaction, C. trachomatis TRPB targeted LAMP can be performed using set 1 primers (Table 3) for additional positive controls (analysis sensitivity is at least 5 C. trachomatis cells per test). Furthermore, using the set 1 primer (Table 3), H. sapiens GAPDH target LAMP could be used as a positive control for the reaction.

Table 3: Specific primer sequences for targets provided in Table 1 for loop-mediated isothermal amplification (LAMP)

Claims (13)

  A method for the detection of nucleic acid targets from biological samples and body fluids, the method comprising: a) cell lysis and sample pretreatment steps comprising the release of nucleic acid targets in biological samples and body fluids; b) nucleic acid amplification Step c) an amplification product detection step, wherein a lytic peptide is used to release a nucleic acid target in a biological sample or body fluid.   2. The method of claim 1, wherein a surfactant is used to release the nucleic acid target in the biological sample or body fluid.   2. The method of claim 1, wherein a combination of lytic peptide and surfactant is used to release a nucleic acid target in a biological sample or body fluid.   2. The method of claim 1, wherein the sequence based on one or more specific targets is amplified.   The method according to claim 1, wherein the sample solution obtained during step (1) is directly subjected to a further amplification procedure.   2. The method of claim 1, wherein qualitative and quantitative detection is performed on the crude sample solution.   2. The method of claim 1, wherein a Chlamydia trachomatis nucleic acid target is detected by use of a specific target region provided in Table 1.   2. The method of claim 1, wherein a Mycoplasma genitalium nucleic acid target is detected by use of a specific target region provided in Table 1.   2. The method of claim 1, wherein the Chlamydia trachomatis nucleic acid target is detected by use of a specific primer and / or a labeled derivative sequence provided in Tables 2 and 3.   2. The method of claim 1, wherein the Mycoplasma genitalium nucleic acid target is detected by use of a specific primer and / or a labeled derivative sequence provided in Tables 2 and 3.   2. The method of claim 1, wherein the human genomic GAPDH target is used for detection as an internal validation and platform evaluation technique.   The method of claim 1, wherein the human genomic GAPDH target is used for detection as an internal validation and platform evaluation technique with specific primers and / or labeled derivative sequences provided in Tables 2 and 3.   A molecular diagnostic method for Chlamydia trachomatis, wherein the TRPB gene is used as a molecular diagnostic target.