File Name: multiplex pcr critical parameters and step by step protocol .zip
One optimal universal primer UP was selected from three UPs by comparing their sensitivities and specificities. All specific primers were tagged with the UP sequence at 5' end, and applied to the multiplex PCR system.
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However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study.
During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.
It is the viral strain that causes coronavirus disease COVID , whose major symptoms include severe pneumonia in humans 2. In addition to pneumonia, other symptoms include high fever, dry cough, fatigue, and frequent pain in the digestive system 3. As of April 11, , COVID affects countries, and the estimated case number has reached 1,, people, with a death toll of over , people, while the number of recovered patients is approximately , people 4.
Due to the spread of the pandemic, there is a desperate need for the development of diagnostic and treatment tools for COVID Most of these kits are based on real-time polymerase chain reaction real-time PCR technology.
The extent of virus transmission during the incubation period is uncertain, but studies have shown that the pharynx shows its peak viral load approximately 4 days after infection 7 , 8. More alarmingly, numerous cases of asymptomatic transmission transmission of the virus by a virus-carrying person before they show any symptoms have been reported 9. R 0 is a measurement of how many people a contagious person transmits the virus to.
However, according to Swedish researchers, the average R 0 is 3. More recently, Sanche et al. The highly infectious nature of SARS-CoV-2 calls for the rapid distribution of detection protocols, diagnostic tools, and treatment options. However, there are many obvious limitations to the current diagnostic methods.
Many companies have developed and sold diagnostic kits hurriedly and hastily as the demand has soared, without proper validation or extensive testing.
In fact, China and the United States reported problems with the reliability of the available test kits at the beginning of the COVID outbreak in each country 13 , In our recent report, we have demonstrated the possibility of obtaining a false-positive result when primers are not verified during the development of primer sets for the detection of SARS-CoV-2 through real-time PCR 9.
A similar report appeared in a medical archive, indicating the possibility of a false positive when the authors compared the nine previously published primer sets for SARS-CoV-2 To make matters worse, many commercially available kits do not publicly disclose the critical sequence information of the primer sets that they contain, making it impossible to carefully verify and validate the quality and sensitivity of each primer set.
In response to the growing need and lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for early detection of the virus in a previous study 9. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results 9.
To address this issue, we set out to develop simple, clear guidelines for designing and optimizing a primer set.
In addition, we expanded the previously developed real-time PCR-based protocol to a more conventional PCR-based protocol and applied a multiplex PCR-based protocol to facilitate the broad availability and easy accessibility of the protocol. The purpose of sampling and the procedure involving the use of a pharyngeal swab were approved by the Seoul National University Hospital Institutional Review Board IRBY-H and thoroughly explained to each volunteer.
We received written consent from the volunteer. A total of one volunteer participated in this experiment Volunteer U. The detailed pharyngeal swab procedure was modified from the previously described procedure 9. Briefly, we used a polyester swab with a plastic shaft 6. After sample collection, the swab was immediately placed in a 1.
HEKT cells are a commonly used human cell line derived from the human embryonic kidney HEK cell line that expresses a mutant version of the SV40 large T antigen The clear upper aqueous layer, which contained RNA, was transferred to a new 1. The Vero cell line was originally isolated from kidney epithelial cells extracted from an African green monkey rhesus macaque In silico primer tests were performed on two websites, idtdna. At idtdna. We performed in silico PCR at the genome.
The primer sets were synthesized and delivered by Cosmogenetech Seoul, Republic of Korea. The primer sets designed and used in this study are listed in Table 1. The accuracy and efficiency of each primer set were verified through PCR amplification of the positive control to optimize the PCR conditions. We used a premixed PCR reagent to minimize the side effects such as contamination. The PCR conditions were the same as those described above. In a previous study, we recognized the importance of the process of verifying and optimizing the primer sets used for virus testing.
Therefore, in this study, we first describe the detailed guidelines for designing and optimizing the primer sets in three important steps Fig. For an optimal target sequence, when there is a splicing variant in a gene transcript, a target region in the more abundant splicing variant is preferred. The second step is the in silico validation of primer and amplicon sequences.
The identification of amplicon secondary structure and the possibility of self- or heterodimer formation by the primer sequence itself were predicted at another website, idtdna. Finally, at the gemone. The third step is to experimentally confirm and optimize the primer set in a biological laboratory.
As a cautious measure, it is important to note that the final concentration of the primer set in the PCR mixture is critical for target-specific PCR. At concentrations greater than the optimal concentration, primers can form dimers and interfere with target-specific PCR. When confirming the PCR result with electrophoresis, the efficiency of the reaction needs to be checked based on whether the band size of the amplicon and the amount of template added are appropriate or not. Step 1: target genes were selected from genomic databases, and primers were designed using Primer3.
Step 2: the primer sets were optimized in silico to avoid secondary structure in the primers or untargeted amplification. Step 3: the designed primers were optimized at the wet-lab level to achieve a high specificity and efficiency for detecting the targets. The first example demonstrated the appearance of spurious primer—dimer formation Fig. A primer dimer is a by-product of PCR, consisting of primer molecules that hybridize with each other because of strings of complementary bases in the primers In fact, primer—dimer bands were found under all conditions, regardless of the presence of the template.
In addition, the intensity of primer—dimer bands tended to decrease as the temperature increased. The appearance of primer—dimer bands suggests that the primer concentration and T a are not optimal and need to be optimized by decreasing the primer concentration and increasing T a. Red asterisks indicate long primer dimers.
The second example demonstrated the appearance of spurious long dimer along with a shorter primer—dimer band Fig. These results suggest that when a primer set shows the potential to form a long PCR by-product, this can be prevented by increasing T a.
The third example revealed a low-efficiency primer set. The resulting electrophoresis data showed that in all conditions, strong spurious long and short primer bands and weak or no expected bands were observed Fig. The appearance of these strong primer bands indicated that the primer set presents an excessive dimerization tendency that is stronger than its tendency to hybridize with the template. These results suggest that this primer set should be discarded, and that a new primer set should be designed and optimized.
The last example demonstrated the appearance of a very long nonspecific band. The resulting electrophoresis data showed no primer—dimer band and a strong expected band in the conditions containing template DNA Fig. The absence of a primer—dimer band indicated that the primer set itself was an optimal primer set.
In summary, primer sets that show primer—dimer formation can be simply optimized by increasing T a , whereas the primer set used in Fig.
When not optimized, spurious primer dimers and by-products that are formed can lead to the appearance of a false-positive curve at high C t values close to 35 cycles and low melting temperatures, regardless of the presence or absence of the template in real-time PCR When designing a new primer set, one can follow the three-step guidelines that we have proposed in Fig.
We designed and optimized the primer sets for each protocol according to the guidelines shown in Fig. The list of the newly designed primer sets as well as the most optimized primer sets from the previous study 9 are shown in Table 1. Among the primer sets that were developed for the real-time PCR-based detection of SARS-CoV-2 in the previous study 9 , some primer sets were re-evaluated and selected on the basis of the design and optimization guidelines presented in Fig.
Using the selected primer sets, we performed traditional PCR, as shown in Fig. The new primer sets for multiplex PCR detection were optimized as shown in Fig. The no-template condition contained no cDNA. Each row represents each primer set. The Y axis represents the normalized reporter value Rn , which was calculated as the fluorescence signal from SYBR Green normalized to the fluorescence signal of a reference dye.
The graphs on the right represent the melting curve plots, which display data collected during a melting curve stage. Peaks in the melting curve may indicate the melting temperature T m of a target or identify nonspecific PCR amplification.
The purple curve is for the no-template condition. The graphs on the left represent amplification plots. The graphs on the right represent melting curve plots. Expected PCR product sizes are indicated in Table 1. The location of each predicted PCR product in the gel is indicated on the right side.
To develop a traditional PCR detection protocol for SARS-CoV-2 that can be easily implemented in any biological laboratory worldwide, we developed a PCR-based protocol using the best primer sets among the previously developed primer sets 9 after the optimization of each primer set according to the guidelines shown in Fig.
This was confirmed with in silico PCR tool as shown in Fig. Taken together, the results demonstrated that we have developed a traditional PCR-based detection protocol for SARS-CoV-2 that should be feasible to use worldwide in any biological laboratory equipped with a conventional PCR machine. However, those primer sets had not been optimized according to the guidelines shown in Fig. Therefore, in this study, we optimized and selected the best primer sets in an attempt to improve the real-time PCR-based protocol.
Scrub typhus, murine typhus, and leptospirosis are widely neglected infectious diseases caused by Orientia tsutsugamushi , Rickettsia typhi , and pathogenic Leptospira spp. Patients usually present with non-specific symptoms and therefore are commonly diagnosed with acute undifferentiated febrile illness. Consequently, patients face delayed treatment and increased mortality.
Advances in Databases and Information Systems pp Cite as. This paper presents a new program to design specific and reliable primers for amplification of exons in a multiplex PCR. The program is composed of two components: a data acquisition component and a data processing component. The data acquisition component automates time—consuming steps of preparing data for the primers design algorithms.
Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has the potential to produce considerable savings in time and effort within the laboratory without compromising on the utility of the experiment.
It can use many different parameters to estimate the compatibility of primers, such as primer—primer interactions, primer—product interactions, difference in melting temperatures, difference in product length and the risk of generating alternative products from the template. A unique feature of the MultiPLX is the ability to perform automatic grouping of large number thousands of primer pairs. The source code of the program is available on request for academic users.
Стратмор медленно повернулся к Сьюзан. Тоже неподвижная, она стояла у дверей шифровалки. Стратмор посмотрел на ее залитое слезами лицо, и ему показалось, что вся она засветилась в сиянии дневного света. Ангел, подумал. Ему захотелось увидеть ее глаза, он надеялся найти в них избавление.
Правда, это было не то прикосновение, какое он рисовал в воображении, представляя себе их первый физический контакт, но все же… Хейл долго с изумлением смотрел на нее, затем медленно повернулся и направился к своему терминалу. Одно ему было абсолютно ясно: распрекрасная Сьюзан Флетчер бьется над чем-то очень важным, и можно поклясться, что это никакая не диагностика. ГЛАВА 28 Сеньор Ролдан восседал за своим столом в агентстве сопровождения Белена, чрезвычайно довольный тем, как умело обошел глупую полицейскую ловушку. Немецкий акцент и просьба снять девушку на ночь - это же очевидная подстава.
Стратмор вздохнул. - У Танкадо наверняка была при себе копия ключа в тот момент, когда его настигла смерть.
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