Nucleic acid sequence combination for monkey pox virus typing detection, kit and use method of kit

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a nucleic acid sequence combination and a kit for monkey pox virus typing detection and a using method of the kit, the kit comprises a PCR primer probe, a buffer solution, Taq enzyme, primer probes and a buffer solution, the Taq enzyme is fully premixed, the primer probe is added into the buffer solution, and the primer probe is added into the buffer solution. The primer probes comprise forward and reverse amplification primers and detection probes respectively aiming at the monkey pox viruses Ia type, Ib type, IIa type and IIb type, and the sequences are respectively shown as SEQ ID NO.1-SEQ ID NO.12. The kit disclosed by the invention can be used for rapidly detecting and identifying the four types of monkey pox viruses at the same time, and has relatively high accuracy, specificity and sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a nucleic acid sequence combination, reagent kit, and method of use for monkeypox virus typing detection. Background Technology

[0002] Monkeypox virus (MPXV) belongs to the genus Orthopoxvirus. The virus particle is brick-shaped, approximately 200–250 nm in diameter. Its genome is linear double-stranded DNA, approximately 197 kb in length, with a GC content of about 33%, and inverted terminal repeats (ITRs) at both ends, encoding approximately 190 non-repetitive open reading frames. Based on phylogenetic and epidemiological characteristics, MPXV is divided into two major evolutionary branches: the Congo Basin branch (branch I, which includes two subbranches, Ia and Ib) and the West African branch (branch II, which also includes two subbranches, IIa and IIb). Branch I is highly virulent, with a human mortality rate of up to 10%; branch II is less virulent, with a mortality rate of <3%. The circulating strains that caused the global outbreak in 2022–2023 all belonged to subbranch IIb, which can be further subdivided into subbranches such as C.1 and C.1.1. Data from my country’s local surveillance in 2023 showed that both imported and secondary cases were mainly C.1.1, and no high-frequency mutations affecting diagnostic targets were detected in the genome, providing a molecular basis for the continued effectiveness of the PCR strategy.

[0003] Currently, monkeypox is classified as a Class B infectious disease in my country and is included in the key monitoring list of the "Quarantine Regulations for Inbound and Outbound Personnel". Both the World Health Organization (WHO) and my country's "Guidelines for the Diagnosis and Treatment of Monkeypox (2023 Edition)" recommend that nucleic acid amplification assay (NAAT) be used as the gold standard for diagnosis in suspected cases, close contacts, and inbound screening samples. Real-time quantitative PCR (qPCR), due to its high sensitivity, strong specificity, and short detection cycle (<2 h), has become a core tool for disease control and clinical laboratories in various countries. However, most existing commercially available kits target conserved genes at the genus level (such as F3L, E9L, B2R), and can only achieve qualitative detection of "orthopoxvirus genus" or "total nucleic acid of monkeypox virus". They cannot distinguish between Clade I and Clade II, which have high pathogenicity differences, nor can they accurately trace the origin of Clade IIa and Clade IIb subclades, making it difficult to meet the needs of port quarantine, clinical treatment, and epidemiological investigations for simultaneous acquisition of typing information.

[0004] Furthermore, the MPXV genome exhibits length polymorphism and single-base insertions / deletions in its terminal ITR region, making traditional single-target detection strategies prone to false negatives due to mutations in the primer-probe binding region. Single-channel detection also fails to identify potentially coexisting smallpox vaccine strains (such as VCV) or laboratory contamination, posing a risk of biosafety misjudgment. Therefore, developing a single-tube multiplex PCR kit that simultaneously performs quadruple detection of monkeypox virus typing while maintaining high sensitivity has become a pressing technological gap in the in vitro diagnostics field. Summary of the Invention

[0005] In response to the problems mentioned in the background art, such as the susceptibility of false negatives in single-target detection, the inability to accurately identify monkeypox virus genotypes, and the insufficient sensitivity and poor specificity of existing fluorescent PCR detection techniques for monkeypox virus, this invention discloses a nucleic acid sequence combination, a kit, and its usage method for monkeypox virus genotyping detection. By specifically designing primers and probes, the sensitivity and specificity of detection are improved.

[0006] The technical solution of the present invention is as follows: A nucleic acid sequence combination for monkeypox virus typing detection, the sequence combination comprising: The first sequence set for detecting monkeypox virus type Ia includes a forward primer as shown in SEQ ID No. 1, a reverse primer as shown in SEQ ID No. 2, and a probe as shown in SEQ ID No. 3; The second sequence set for detecting monkeypox virus type Ib includes a forward primer as shown in SEQ ID No. 4, a reverse primer as shown in SEQ ID No. 5, and a probe as shown in SEQ ID No. 6; The third sequence group for detecting monkeypox virus type IIa includes a forward primer as shown in SEQ ID No. 7, a reverse primer as shown in SEQ ID No. 8, and a probe as shown in SEQ ID No. 9; The fourth sequence group for detecting monkeypox virus type IIb includes a forward primer as shown in SEQ ID No. 10, a reverse primer as shown in SEQ ID No. 11, and a probe as shown in SEQ ID No. 12.

[0007] Furthermore, the probe sequence shown in SEQ ID No. 3 is labeled with a CY5 fluorescent group at its 5' end and a BHQ1 fluorescent quencher group at its 3' end; The probe sequence shown in SEQ ID No. 6 has a ROX fluorescent group labeled at the 5' end and a fluorescence quencher group BHQ1 labeled at the 3' end; The probe sequence shown in SEQ ID No. 9 has a VIC fluorescent group labeled at the 5' end and a fluorescence quenching group MGB labeled at the 3' end; The probe sequence shown in SEQ ID No. 12 has a FAM fluorescent group labeled at the 5' end and a fluorescence quencher group MGB labeled at the 3' end.

[0008] Furthermore, the primer sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 10, and SEQ ID No. 11 are modified with locked nucleic acids.

[0009] Furthermore, the locked nucleic acid modification position in the primer sequence shown in SEQ ID No. 1 is the 5th position at the 3' end; The locked nucleic acid modification position in the primer sequence shown in SEQ ID No. 2 is the 4th position at the 3' end; The locked nucleic acid modification position in the primer sequence shown in SEQ ID No. 4 is the 5th position at the 3' end; The locked nucleic acid modification position in the primer sequence shown in SEQ ID No. 5 is the 3rd position at the 3' end; The locked nucleic acid modification position in the primer sequence shown in SEQ ID No. 7 is the 4th position at the 3' end; The locked nucleic acid modification position in the primer sequence shown in SEQ ID No. 8 is the 3rd position at the 3' end; The locked nucleic acid modification position in the primer sequence shown in SEQ ID No. 10 is the 4th position at the 3' end; The locked nucleic acid modification position in the primer sequence shown in SEQ ID No. 11 is the 3rd position at the 3' end.

[0010] The present invention also provides a kit for monkeypox virus typing detection, the kit comprising the nucleic acid sequence combination for monkeypox virus typing detection as described above, the kit being used to detect monkeypox virus type Ia, monkeypox virus type Ib, monkeypox virus type IIa, and monkeypox virus type IIb.

[0011] Furthermore, the kit is a fully premixed reagent, and the kit also includes a premixed solution and RNase-free water, wherein the premixed solution includes a buffer solution and a Taq enzyme mixture.

[0012] Furthermore, the final concentration of the primers in the reagent is 400-500 nM, and the final concentration of the probe is 200-300 nM.

[0013] This invention also provides a method for using a kit for monkeypox virus typing detection, the specific steps of which include: Step 1: Extract nucleic acid from the sample to be tested; Step 2: Thaw and mix the reagents in the kit, then aliquot them and add them to the extracted test sample, negative control, and positive control into PCR reaction tubes respectively. Place the PCR reaction tubes into the real-time PCR device, set the reaction conditions, and perform real-time PCR detection. Step 3: After the reaction in Step 2 is completed, set up automatic baseline adjustment and interpret the test results as positive, doubtful, or negative based on the amplification curve and Ct value.

[0014] Furthermore, the reaction conditions are: pre-denaturation stage 94℃, 2 min; amplification stage 94℃, 10 s, 56℃, 50 s, 40 cycles.

[0015] Furthermore, the criteria for interpreting a positive result in step three are: the appearance of an "S"-shaped amplification curve and a Ct value ≤ 35; The criteria for interpreting a suspicious result are: the appearance of an "S"-shaped amplification curve and a Ct value > 35; The criteria for interpreting a negative result are: no Ct value or the amplification curve exceeds the threshold but does not show an "S" shape.

[0016] Compared with the prior art, the kit of the present invention has the following advantages: (1) This invention enables the detection and identification of four subtypes of monkeypox virus, accurately identifying the specific pathogen, guiding further treatment, and improving the scientific rigor and effectiveness of treatment plans. Identification of the specific pathogen helps in the statistical analysis of the type and prevalence of the infecting pathogen, understanding the detection rate and prevalence trend of the infecting pathogen, and thus taking scientific preventative measures. Furthermore, compared to other detection kits currently on the market, the kit provided in this invention has a detection limit as low as 500 copies / mL for monkeypox virus, significantly improving detection sensitivity.

[0017] (2) This kit uses a fully premixed solution, that is, the primers, probes, buffer and Taq enzyme are premixed into one tube. When using, you only need to thaw, mix and dispense, which eliminates the step of preparing the reaction system and greatly improves the work efficiency. In addition, the amplification program of this invention has been optimized and the fluorescence acquisition time has been optimized, which greatly shortens the detection reaction time and can shorten the detection time to within 1 hour.

[0018] (3) The kit provided in this invention uses locked nucleic acid modification on the primers for specific sites, which improves the amplification efficiency of the primers and further enhances the detection sensitivity while ensuring specificity. Compared with primer sequences without locked nucleic acid modification, the detection sensitivity of this invention is improved by about 5 times. Attached Figure Description

[0019] Figure 1 The image shows the detection results of monkeypox virus type Ia, monkeypox virus type Ib, monkeypox virus type IIa and monkeypox virus type IIb using a kit for monkeypox virus typing detection in an embodiment of the present invention. Figure 2 This is a graph showing the experimental results of using the primers and probes of this invention to detect the target sequences of various detection indicators in Experiment Example 1 of this invention; Figure 3 A comparison of the detection results of monkeypox virus type Ia samples using the locked nucleic acid modified primer system and the conventional primer system in Experiment Example 2 of this invention; Figure 4 A comparison of the detection results of monkeypox virus type Ib samples using the locked nucleic acid modified primer system and the conventional primer system in Experiment Example 2 of this invention; Figure 5 A comparison of the detection results of monkeypox virus type IIa samples using the locked nucleic acid modified primer system and the conventional primer system in Experiment Example 2 of this invention; Figure 6 A comparison of the detection results of monkeypox virus type IIb samples using the locked nucleic acid modified primer system and the conventional primer system in Experiment Example 2 of this invention; Figure 7 This is a graph showing the cross-reaction detection results of the primer and probe detection system of this invention with five other pathogens in Example 4 of this invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0021] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0022] This embodiment provides a nucleic acid sequence combination, a reagent kit, and its usage method for monkeypox virus typing detection. The detection kit is prepared using a 25 μL amplification reaction system as an example, and the preparation scheme is shown in Table 1 below: Table 1: Reagent Preparation Table The premixed solution was a mixture of buffer solution and Taq enzyme, purchased from Novizan, product name: PureAmp U+Super Multiple Probe qPCR PreMix (Low DNA), catalog number: UQN221-C1. The primer and probe names and their corresponding sequences in Table 1 are shown in Table 2 below. Table 2: Primer and probe sequences and positions of nucleic acid modifications in this invention In the primer sequences in the table above, the specific bases “A+”, “C+” and “G+” are locked nucleic acid modified bases. The primer sequences used to detect monkeypox virus type Ia are shown in SEQ ID No. 1 and SEQ ID No. 2, and the detection probe sequence is shown in SEQ ID No. 3. Its 5' end is labeled with the CY5 fluorescent group, and its 3' end is labeled with the BHQ1 fluorescent quencher group. The primer sequences for detecting monkeypox virus type Ib are shown in SEQ ID No. 4 and SEQ ID No. 5, and the detection probe sequence is shown in SEQ ID No. 6. The 5' end is labeled with the ROX fluorescent group, and the 3' end is labeled with the BHQ1 fluorescent quencher group. The primer sequences for detecting monkeypox virus type IIa are shown in SEQ ID No. 7 and SEQ ID No. 8, and the detection probe sequence is shown in SEQ ID No. 9. The 5' end is labeled with a VIC fluorescent group, and the 3' end is labeled with a fluorescence quencher group MGB. The primer sequences for detecting monkeypox virus type IIb are shown in SEQ ID No. 10 and SEQ ID No. 11, and the detection probe sequence is shown in SEQ ID No. 12. The 5' end is labeled with a FAM fluorescent group, and the 3' end is labeled with a fluorescence quencher group MGB.

[0023] This embodiment also provides a method for using the above-mentioned test kit, including the following steps: Step 1: Extract nucleic acid from the sample to be tested; Specifically, nucleic acids were extracted from positive samples of monkeypox virus type Ia, monkeypox virus type Ib, monkeypox virus type IIa, and monkeypox virus type IIb for the verification of the detection system. The indicators of each positive sample were confirmed by sequencing.

[0024] Step 2: Thaw and mix the reagents in the kit, then aliquot them and add them to the extracted test sample, negative control, and positive control into PCR reaction tubes respectively. Place the PCR reaction tubes into the real-time PCR device, set the reaction conditions, and perform real-time PCR detection. Specifically, after vortexing the prepared reagents, centrifuge briefly and then directly aliquot 20 μL into each well of a 96-well plate or eight-tube strip. Take the aliquoted reagents to the sample loading chamber and add 5 μL each of the pre-extracted sample nucleic acid, negative control, and positive control to different PCR reaction tubes. Cap the tubes or seal them, centrifuge briefly, and then take them to the amplification chamber for instrumental testing.

[0025] Step 3: After the reaction in Step 2 is completed, set up automatic baseline adjustment and interpret the test results as positive, doubtful, or negative based on the amplification curve and Ct value.

[0026] Specifically, the reaction conditions were set as follows: pre-denaturation stage 94℃ 2min; amplification stage 94℃ 10s, 56℃ 30s, cycled 40 times. After setting, the detection program was run. The interpretation criteria for the amplification curve and Ct value results are as follows: Positive: An "S"-shaped amplification curve appears, and the Ct value is ≤35; Suspicious: An "S"-shaped amplification curve appears, but the Ct value is >35; Negative: No Ct value; or the curve exceeds the threshold but does not show an "S" shape. For doubtful results, the experiment should be repeated. If the repeated experiment shows an "S" shaped amplification curve and the negative control is uncontaminated, it can be judged as positive.

[0027] The experimental results are attached. Figure 1 As shown in the results, the detection kit of the present invention can accurately detect the nucleic acids of monkeypox virus type Ia, monkeypox virus type Ib, monkeypox virus type IIa, and monkeypox virus type IIb samples simultaneously, and all samples exhibit good linearity.

[0028] Experimental Example 1: Detection Limit Experiment Experimental Methods: Reference samples for monkeypox virus types Ia, Ib, IIa, and IIb were tested according to the detection kit and method provided in this embodiment of the invention. The reference samples were artificially synthesized, precisely quantified nucleic acid samples. After serial dilution, solutions at concentrations of 5000 copies / mL, 2500 copies / mL, 1000 copies / mL, 500 copies / mL, and 250 copies / mL were selected for instrumental detection to verify the limit of detection and linearity of the kit.

[0029] The experimental results are attached. Figure 2It is known that when using the primers and probes of the present invention to detect the detection limit reference of each indicator, the detection system exhibits good linearity. The lowest detection limit of each indicator of the present invention can reach 500 copies / mL, while the lowest detection limit of similar multiplex detection kits on the market or similar multiplex detection methods reported in the literature claims to be 1000 copies / mL. Detection using the primers and probes of the present invention has superior sensitivity.

[0030] Experiment Example 2: The effect of locked nucleic acid modification on detection sensitivity Experimental Methods: Following the detection kit and method provided in the embodiments of this invention, conventional primers without locked nucleic acid modification were set as control group 1. Monkeypox virus types Ia, Ib, IIa, and IIb were detected respectively. The samples were identical to those in the detection kits described in the embodiments. This experimental example detected nucleic acids from positive samples of each indicator at a concentration of 1000 copies / mL to verify whether locked nucleic acid modification can effectively improve the detection sensitivity of the detection kit of this invention.

[0031] The experimental results are shown in Table 3 below. As can be seen from the table, the nucleic acid-modified primer system used in this invention can detect the Ct values ​​of each indicator channel 2-3 times earlier than the control group primer system 1, reflecting that the detection sensitivity of this invention is about 5 times higher than that of the control group 1. At the same time, the signal values ​​of this invention are also significantly improved; the signal values ​​of the modified primer system are distributed between 850-2600, while the signal values ​​of the conventional primer system are between 500-2300.

[0032] Table 3: Comparison of detection results between the primer system of this invention and the primer system without locked nucleic acid modification. Experiment Example 3: The Influence of Probe Sequence on Amplification Signal Values Experimental Methods: Following the detection kit and method provided in this embodiment of the invention, the probe sequence used was replaced to form control group 2. Monkeypox virus types Ia, Ib, IIa, and IIb were detected respectively to verify the specificity of the probe sequence used in this invention. The specific probe sequence information of control group 2 is shown in Table 4 below: Table 4: Probe sequence listing for control group 2 Comparative experiments were conducted between Example 1 and Comparative Example 2. The reaction system was prepared as in Example 1, and the template loading volume was 5 μL. The template was a target sequence solution of each indicator, with a concentration of 2500 copies / μL. Detection was performed using an ABI 7500 fluorescence PCR instrument, and the differences in Ct values ​​and signal values ​​between Example 1 and Comparative Example 2 were compared. The experimental results are shown in Table 5 below: Table 5: Detection Results of Example 2 and Control Group 2 index Example signal value Control group 2 signal value Signal value difference Monkeypox virus type Ia 2335 2013 322 Monkeypox virus type Ib 2064 1562 502 Monkeypox virus type IIa 2122 1803 319 Monkeypox virus type IIb 1971 1410 561 Analysis of experimental results: As shown in Table 5, the probe sequence selected in this invention is superior to control group 2 in terms of signal value. A higher signal value can indirectly improve sensitivity. At the same time, a higher signal value results in a more standard amplification curve, making it easier to interpret results when detecting low-concentration samples. In addition, it has better specificity.

[0033] Example 4: Specificity test of the primer-probe detection system of the present invention Experimental Methods: Following the detection kit and method provided in this invention, five other pathogen cultures or nucleic acid samples with similar symptoms, infection sites, or species to the pathogens identified by this reagent were selected. The specificity of the detection kit provided by this invention for monkeypox virus type Ia, monkeypox virus type Ib, monkeypox virus type IIa, and monkeypox virus type IIb was tested. These five indicators include: vaccinia virus, herpes simplex virus, varicella-zoster virus, Staphylococcus aureus, and Candida albicans.

[0034] The experimental results are attached. Figure 7 As shown. From the appendix Figure 7 As can be seen from this, the detection kit provided by the present invention has no cross-reaction with the above 5 pathogens and has good specificity.

Claims

1. A combination of nucleic acid sequences for monkeypox virus typing, characterized in that, The sequence combination comprises: a first sequence group for detecting monkeypox virus type Ia, comprising a forward primer as shown in SEQ ID No. 1, a reverse primer as shown in SEQ ID No. 2 and a probe as shown in SEQ ID No. 3; a second sequence group for detecting monkeypox virus type Ib, comprising a forward primer as shown in SEQ ID No. 4, a reverse primer as shown in SEQ ID No. 5 and a probe as shown in SEQ ID No. 6; a third sequence group for detecting monkeypox virus type IIa, comprising a forward primer as shown in SEQ ID No. 7, a reverse primer as shown in SEQ ID No. 8 and a probe as shown in SEQ ID No. 9; a fourth sequence group for detecting monkeypox virus type IIb, comprising a forward primer as shown in SEQ ID No. 10, a reverse primer as shown in SEQ ID No. 11 and a probe as shown in SEQ ID No.

12.

2. The combination of nucleic acid sequences for monkeypox virus typing detection according to claim 1, characterized in that: The probe sequence as shown in SEQ ID No. 3 is labeled with a CY5 fluorescent group at the 5' end and a fluorescent quenching group BHQ1 at the 3' end; The probe sequence as shown in SEQ ID No. 6 is labeled with a ROX fluorescent group at the 5' end and a fluorescent quenching group BHQ1 at the 3' end; The probe sequence as shown in SEQ ID No. 9 is labeled with a VIC fluorescent group at the 5' end and a fluorescent quenching group MGB at the 3' end; The probe sequence as shown in SEQ ID No. 12 is labeled with a FAM fluorescent group at the 5' end and a fluorescent quenching group MGB at the 3' end.

3. The combination of nucleic acid sequences for monkeypox virus typing detection according to claim 1, wherein, The primer sequences as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. 11 are modified with a locked nucleic acid.

4. The combination of nucleic acid sequences for monkeypox virus typing according to claim 3, characterized in that: The locked nucleic acid modification position in the primer sequence as shown in SEQ ID No. 1 is the 5th position at the 3' end; The locked nucleic acid modification position in the primer sequence as shown in SEQ ID No. 2 is the 4th position at the 3' end; The locked nucleic acid modification position in the primer sequence as shown in SEQ ID No. 4 is the 5th position at the 3' end; The locked nucleic acid modification position in the primer sequence as shown in SEQ ID No. 5 is the 3rd position at the 3' end; The locked nucleic acid modification position in the primer sequence as shown in SEQ ID No. 7 is the 4th position at the 3' end; The locked nucleic acid modification position in the primer sequence as shown in SEQ ID No. 8 is the 3rd position at the 3' end; The locked nucleic acid modification position in the primer sequence as shown in SEQ ID No. 10 is the 4th position at the 3' end; The locked nucleic acid modification position in the primer sequence as shown in SEQ ID No. 11 is the 3rd position at the 3' end.

5. A kit for monkeypox virus typing, characterized by: The kit comprises the nucleic acid sequence combination for monkeypox virus typing detection as claimed in any one of claims 1-4, and the kit is used for detecting monkeypox virus type Ia, monkeypox virus type Ib, monkeypox virus type IIa and monkeypox virus type IIb.

6. The kit for the detection of monkeypox virus typing according to claim 5, characterized in that: The kit further comprises a premixed solution and RNase-free water, wherein the premixed solution comprises a buffer solution and a mixed solution of taq enzyme.

7. The kit for detection of monkeypox virus typing according to claim 5, characterized in that: The final concentration of the primer sequence in the nucleic acid sequence combination of the kit is 400-500 nM, and the final concentration of the probe sequence is 200-300 nM.

8. The use of a kit for typing monkeypox virus according to any one of claims 5 to 7, characterized in that, The kit comprises the following steps: Step one: extracting nucleic acid of the sample to be detected; Step two: after the reagents in the kit are thawed and mixed, they are divided into portions, and the extracted sample to be detected, negative control and positive control are added into PCR reaction tubes respectively, the PCR reaction tubes are placed into a fluorescent quantitative PCR device, and reaction conditions are set to perform fluorescent PCR detection; Step three: after the detection reaction in step two is completed, an automatic baseline adjustment is set, and the detection results are interpreted according to the amplification curve and Ct value to obtain positive results, suspicious results and negative results.

9. The method of using the kit for the detection of monkeypox virus typing as claimed in claim 8, wherein: The reaction conditions in step two are pre-denaturation at 94℃ for 2 min and amplification at 94℃ for 10 s and at 56℃ for 30 s, with 40 cycles.

10. The method of using the kit for typing monkeypox virus as claimed in claim 8, wherein: The positive result interpretation standard in step three is that an "S" type amplification curve appears and the Ct value is less than or equal to 35; The suspicious result interpretation standard is that an "S" type amplification curve appears and the Ct value is greater than 35; The negative result interpretation standard is that there is no Ct value or the amplification curve exceeds the threshold value but is not "S" type.

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