Tuberculosis is a re-emerging infectious disease that causes death by outbreak (1).
DNA based amplification reactions and antigen-antibody based methods are available for the diagnosis of tuberculosis except for the culture method which is the gold standard (7). However, since the diagnosis of the disease takes a long time with the culture method, treatment is delayed. Also, because of the low sensitivity of the antigen-antibody based methods, it is a needed to the rapid and sensitive method for diagnosis of tuberculosis. Although nucleic acid based diagnostic systems are advantageous in terms of time and sensitivity, many of them have complicated protocols and depend on complex devices (8).
Molecular methods, especially PCR, have reached an important point for the diagnosis of tuberculosis (9). In addition to the commercially tuberculosis PCR detection kits, many laboratories have their own in-house methods (10).
However, PCR methods used in routine require specialists due to their complex processes and cold-chain transportation needs. Generally, PCR studies are performed with small volumes such as 1-10 μl and the smallest error can have critical results (11). Therefore, the method to be developed should be as low as possible to minimize the person's error.
In recent years, scientists have been working on the use of DNA amplification reactions in the point of care tests (12). The loop-mediated isothermal amplification (LAMP) method developed by Notomi et al. (2000) is more sensitive than other PCR methods, because of using 4 or 6 different primers that recognize 6 or 8 different regions on the target gene (13, 14). Due to its primers design and Bst DNA polymerase, the method does not require temperature cycling (15). Whereby its ability to perform at a constant temperature, it has become frequently used in point of care tests and microfluidic chips (16, 17). Particularly, the fact that the detection of the products formed at the end of the reaction can be made with the naked eye or the lateral flow test strips makes it widely used in field studies (18).
Despite the advantages of using DNA amplification methods for diagnostic purposes, there are still challenges that need to be overcome (19). Standardization of reaction mixture preparation is important for the accuracy and sensitivity of the results. It is difficult to ensure standardization, especially in field conditions, as the ingredients must be stored at -20 °C and work in the cold chain. Lyophilization is a protection method used in amplification reactions to make the mixtures resistant to environmental conditions (20). The lyophilization process, which is also used in commercial kits, extends the lifetime of PCR mixtures up to 1 year (21).
The aim of this study was to extend lifetime and resistant to environmental conditions of LAMP and RT-PCR mixtures via lyophilization. Accordingly, three different lyophilization methods were tried and results were compared.
The specificity, sensitivity, positive predictive value and negative predictive value were determined from the results based on a calculation previously reported (23).
Two different cryoprotectant mixtures which described by Sharma et al. (2014) and Klatser et al. (1998) (20, 24), except our mixture was used for LAMP and RT-PCR methods. The tested lyophilization contents in this study are given in table 1. Lyophilization was performed on the Christ Epsilon 2-6D LSCplus freeze dryer and the protocol is given in table 2. Amplification reactions were performed on a lyophilized mixture with 20 μl of molecular grade water and 5 μl of DNA sample (Tab. 1 and Tab. 2).
Cryoprotectant mixtures and amount of ingredients
The protocol of freeze dryer
The lyophilized LAMP and RT-PCR mixtures were stored at – 20 °C, 4 °C and room temperature to reveal the thermal stability of lyophilized mixtures. Three sets of vials were used from each of the stored temperatures. Reaction mixtures were used for amplification on 1st, 2nd, 4th weeks and 2nd, 6th and 12th months.
Serial dilutions between 105 CFU/ml and 101 CFU/ml of
For the lyophilization of LAMP and RT-PCR mixtures, three different cryoprotectant mixtures were tested. In the studies carried out as a result of the lyophilization process, described by Sharma et al., (2014) and Klatser et al., (1998) (20, 24) contents were found to cause inhibition in both amplification reactions which can be shown in figure 2. The cryoprotectant mixture tested for the first time in this study is suitable for LAMP and RT-PCR studies. Accordingly, the cryoprotectant given in table 1 was tested at the indicated concentrations. As shown in figure 2, the cryoprotectant mixture proposed by Sharma et al., (2014) and Klatser et al., (1998) (20, 24) has not been successful in our studies (Fig. 1 and Fig. 2).
Lyophilized mixtures were stored at – 20 °C, 4 °C and room temperature to protect from light. Lyophilized samples were tested in 3 replicates, both positive and negative, at 1, 2, 4 weeks, 2, 6 and 12 months. No inhibition was observed at – 20 °C and 4 °C conditions for 12th month. However, the lyophilized samples stored at room temperature did not work after 2 weeks (Fig. 3).
Serial dilutions between 105 CFU/ml and 101 CFU/ml of the
The culture method is still the gold standard for the diagnosis of tuberculosis (25). However, in recent years, molecular tests have been studied for rapid diagnosis in the field. Lyophilization of the master mixes is an indispensable requirement for the developed molecular tests to be used in the field. There are different stabilizers used for this purpose such as trehalose, PEG, Stachyose, glycerol (12, 26). However, the stabilizers to be preferred must be compatible with the molecular method used. For the lyophilization of LAMP and RT-PCR mixtures, three different cryoprotectant mixtures were tested. In the study of Sharma et al., (2014) (24), the multiplex RT-PCR method was developed for the diagnosis of foot and mouth disease virus (FMDV) and the optimization and validation of the lyophilization of the reaction content was performed. But, the cryoprotectant mixture proposed by Sharma et al., (2014) (24), has not been successful in our studies.
Undoubtedly, diagnostic mixes developed as lyophilized have a longer life than non-lyophilized mixes (27). Nevertheless, even if they are lyophilized, these mixtures have certain periods in which they can maintain stability. The stability of the lyophilization developed by this study was evaluated up to 12 mounth at 3 different temperatures (– 20 °C, 4 °C and room temperature). All lyophilized samples were assayed with 3 replicates and positive and negative samples. Studies were performed at the 1st, 2nd, 4th weeks and 2nd, 6th and 12th month after lyophilization. Any inhibition was not observed at – 20 °C and 4 °C conditions for 12 month but the lyophilized samples stored at room temperature did not work after 2nd weeks. Chua et al. (2011) reported that the lyophilized PCR mixture remains stable for 7.4 months at 24 °C. But unlike our study, the thermal stability of the lyophilized PCR assay was estimated using accelerated aging techniques with elevated temperatures also known as the Q10 method in Chua et al., (2011) study. Furthermore, the thermal stabilization of the lyophilized amplification mixture determined at room temperature for 2 weeks is enough for field studies. In a different study, the stability of the lyophilized mixture was studied at 3 different temperatures (4 °C, 25 °C and 37 °C), over 30 days (28). According to Khazani et al., (2017), the lyophilized PCR mixture was stable for 30 days. Here, it can be said that the type and amount of cryoprotectants used may be important to the stabilization of mixtures.
The infectious dose of many infectious microorganisms is quite low (29). It is very important to determine the infective dose of these pathogens with the methods to be developed for the patient’s health. For this purpose, the LOD is determined during the kit development stages. In this study, determination of the limit of detection of the lyophilized master mix was performed. As a result, the detection limit of the lyophilized LAMP master mix was 102 CFU/ml and the RT-PCR master mix was 101 CFU/ml. Here, it is seen that the lyophilization process does not affect the detection limit of both two methods. In the lyophilization study of the PCR mixture used in the detection of S. aureus by Nagaraj et al. (2018) (26), the limit of detection was determined as 106 CFU/mL. Although the limits of detection are different in the studies, especially, parallel results between lyophilized and fresh mixtures demonstrate an important advantage of lyophilization processes.
In conclusion, this study provides a suitable lyophilization process for LAMP and RT-PCR mixtures used in the detection of